Water-Activation Steaming Coupled with Single-Marker Quantification: A Green Strategy for Industrial Standardization of Bioactive Steamed Panax notoginseng​

Water-Activation Steaming Coupled with Single-Marker Quantification: A Green Strategy for Industrial Standardization of Bioactive Steamed Panax notoginseng​ | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Water-Activation Steaming Coupled with Single-Marker Quantification: A Green Strategy for Industrial Standardization of Bioactive Steamed Panax notoginseng​ Ning Yifei, Wang Nan, Tian Shaoqiong, Liang Yinxiong, Ma Ji, Cui Xuiming This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7517118/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Mar, 2026 Read the published version in BMC Chemistry → Version 1 posted 12 You are reading this latest preprint version Abstract Industrial production of Steamed Panax notoginseng (SPN) faces batch-to-batch variability (RSD > 15%) in rare ginsenosides Rk 3 , Rh 4 , 20( S )-Rg 3 , and 20( R )-Rg 3 and costly quality control due to expensive reference standards (e.g., 20( S )-Rg 3 ≈ $ 2500/mg). To address this, we developed an integrated strategy: (1) A "water-activation–gradient temperature control" process optimized via orthogonal design and Arrhenius kinetics (Ea = 58.3 kJ/mol) increased total rare ginsenosides by 78.6% (32.7 mg/g, p < 0.01) under the following optimized parameters: particle size of 2 ~ 4 mm, water impregnation of 100% w/w for 2 h, and steaming at 120°C for 5 h, This optimization reduced batch variability to an RSD < 5%; (2) An HPLC-QAMS method using accessible 20( R )-Rg 3 as an internal reference achieved simultaneous quantification of four ginsenosides with validated relative correction factors (Rk 3 : 0.5768, Rh 4 : 0.4690, 20( S )-Rg 3 : 1.0924; RSD < 2.0%), demonstrating high accuracy (recovery: 91.95–101.34%, RSD < 1.8%), linearity (r ≥ 0.9997), and robustness across HPLC systems (RSD < 3.5%), reducing reference standard costs by 75%. The QAMS method exhibited superior greenness (AGREE score: 0.76 vs. 0.63 for ESM) and applicability (BAGI score: 77.5 vs. 65.0). Analysis of 15 batches confirmed consistency (RE% < 5% vs. ESM), while optimized extraction (60% ethanol, 5 cycles × 1.5 h) achieved 85.82% transfer rate for 20( R )-Rg 3 . This work resolves SPN industrialization bottlenecks by ensuring bioactive consistency and establishing a cost-effective, eco-friendly quality control model transferable to other processed botanicals. Steamed Panax notoginseng Rare ginsenosides Processing optimization QAMS Green analytical chemistry Industrial standardization AGREE BAGI Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Panax notoginseng (Burk.) F.H.Chen, a prized medicinal herb in the Araliaceae family, has been extensively applied in traditional Chinese medicine (TCM) with a documented history tracing back to the Compendium of Materia Medica ( Bencao Gangmu ) in the Ming Dynasty. Renowned for the principle of "raw for dispersion, processed for tonification" , its raw form dissipates stasis and arrests bleeding, while the steamed form tonifies vitality and strengthens the body—exemplifying the TCM theory of differential treatment based on processing state ( Sheng Shu Yi Zhi )[1]. Modern pharmacological studies have confirmed that the primary bioactive constituents of P. notoginseng are saponins , with over 150 identified types. These include two main categories: (1) Protopanaxadiol - type (PPD - type) saponins (e.g ., ginsenosides Rb 1 , Rd);and (2) Protopanaxatriol -type (PPT-type) saponins (e.g., ginsenosides Rg₁, Re)[2-5]. These compounds exert hemostatic , anti-inflammatory , and cardiovascular protective effects by modulating key signaling pathways such as PI3K/AKT and NF-κB [6-8] . Chemical Transformations During Processing of Raw Panax notoginseng and Their Pharmacological Implications The thermal processing of raw Panax notoginseng (Burk.) F.H.Chen triggers a series of chemical transformations that fundamentally alter its pharmacodynamic basis and pharmacological actions , establishing the material foundation for the characteristic "differential use of crude and processed forms" . High-temperature processing catalyzes deglycosylation , dehydration , and isomerization of primary saponins, generating rare ginsenosides with enhanced or unique bioactivities[9-11]. Among these, ginsenosides Rk 3 , Rh 4 , 20( S )-Rg 3 , and 20( R )-Rg 3 are established as critical quality markers (CQMs) for SPN, with the following scientific rationale: Thermal Transformation Specificity: These four saponins are the primary degradation products formed via specific pathways under high-temperature/high-humidity steaming (e.g., 105°C, 2~4 h)[12].Their generation follows well-defined routes: Rb 1 →Rd→Rg 3 →Rh 4 ; Rg 1 →Rh 1 →PPT [13]. Their concentrations directly correlate with processing intensity and efficiency (e.g., Rk 3 content increases 4.7-fold after optimal steaming); Distinct Pharmacological Activities : Each rare ginsenoside contributes uniquely to tonifying effects : 20( S )-Rg 3 exhibits potent anti-tumor activity by inducing apoptosis via AKT/mTOR inhibition (IC₅₀=12.3μM). 20( R )-Rg 3 demonstrates neuroprotective effects , increasing neuron survival by 37.2% in Aβ₂₅₋₅₅-induced injury models[14]. Rk₃/Rh₄ synergistically enhance anti-inflammatory , antioxidant , and hematopoietic activities by activating the PI3K/Akt-Nrf2 axis (↓MDA 51%, ↑SOD 73%)[9, 12]; Industrial Quality Control (QC) Bottlenecks : Analytical reference standards for these saponins—particularly epimers 20( S )-Rg 3 and 20( R )-Rg 3 are prohibitively expensive (e.g., 20( S )-Rg 3 ≈ $2,500/mg)[15]. This imposes severe constraints on industrial-scale QC when employing external standard methods (ESM) , which require individual reference compounds. The cost significantly limits batch-monitoring frequency and process scalability . Despite the increasingly clarified material basis for the pharmacological effects of SPN, its industrialization remains constrained by two major technical bottlenecks: Process Variability: Traditional processing parameters—including particle size, water immersion conditions, steaming temperature, and steaming time—lack systematic optimization based on the kinetics of saponin conversion. Studies indicate that conversion rates are highly sensitive to these parameters[16]. For instance, reducing the particle size from 8 mm to 2 mm increases the yield of Rk3 by 2.2-fold. However, excessive pulverization risks thermal degradation of sensitive constituents. Inadequate water immersion fails to fully activate the conversion pathway [17], while suboptimal temperature-time combinations lead to either incomplete conversion or degradation. These inconsistencies directly result in significant batch-to-batch variations (RSD >15%) in the total content of key rare saponins (e.g., Rk 3 , Rh 4 , 20( S )-Rg 3 , 20( R )-Rg 3 ), critically impacting product efficacy and safety ; Limitations of Current Analytical Methods: Existing quality control (QC) primarily relies on the External Standard Method (ESM) for single-component quantification. Although accurate, ESM becomes economically impractical for simultaneously monitoring multiple rare saponins (Rk 3 , Rh 4 , 20( S )-Rg 3 , 20( R )-Rg 3 , etc.) due to the prohibitively high cost and limited availability of individual reference standards. The Quantitative Analysis of Multi-components by Single Marker (QAMS) approach offers a promising alternative [18, 19]. QAMS quantifies multiple analytes using relative correction factors (RCFs) derived from a single reference standard. However, its application to SPN remains unexplored. A core challenge for QAMS lies in ensuring the robustness and transferability of RCF values. These values are susceptible to significant drift (10%) under variations in chromatographic conditions (e.g., column type). HPLC instrument model, detection wavelength, flow rate), thereby compromising method reliability[20]. To address the aforementioned challenges, this study aims to: Optimize the Steaming Process: By integrating orthogonal design with kinetic modeling (based on the identified activation energy for saponin conversion, E a ≈ 58.3 kJ/mol )[13], we will develop a stable, efficient, and scalable " moisture activation-gradient temperature control " production process for SPN. This process targets the maximization of key rare saponin yields (Rk 3 , Rh 4 , 20( S )-Rg 3 , 20( R )-Rg 3 ) while minimizing thermal degradation risks associated with conventional methods. Establish a Robust QAMS Method: We will develop and validate a novel, cost-effective, and reliable HPLC-QAMS method using the readily accessible 20( R )-Rg 3 as the internal reference substance for simultaneous quantification of four target rare saponins in SPN and its extracts. The study will focus on rigorous evaluation of relative correction factor (RCF) stability under varied chromatographic conditions (e.g., column type, flow rate, detection wavelength) to ensure method robustness and transferability. Furthermore, the greenness and environmental sustainability of the proposed method will be comprehensively assessed using Analytical GREEnness (AGREE) and Blue Applicability Grade Index (BAGI) metrics. These tools will generate quantitative scores based on 12 environmental impact criteria (AGREE) and operational practicality indicators (BAGI), enabling objective comparison with conventional methods. This integrated strategy addresses two core challenges-process reproducibility and analytical cost-effectiveness-by establishing a scientifically robust and industrially via ble protocol for the quality standardization of SPN. Consequently, it supports broader clinical and industrial applications while actualizing the value of the "differences in raw and processed products efficacy" theory through modern quality control frameworks. Materials and methods Reagents, Reference Standards and Sample Chromatography-grade solvents (methanol and acetonitrile, HPLC grade) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Ultrapure water was prepared using a Milli-Q system (Millipore, Bedford, MA, USA) from purified water sourced from Wahaha Group (Hangzhou, China). Ginsenoside reference standards : Rk 3 and Rh 4 were obtained from Stanford Analytical Chemicals Inc. (Stanford, CA, USA); 20( S )-Rg 3 was supplied by Chengdu GLP Biotechnology Co., Ltd. (Chengdu, China);20( R )-Rg 3 was procured from the National Institutes for Food and Drug Control (NIFDC, Beijing, China). Purity validation : All standards were verified by HPLC peak area normalization , with purities exceeding 98% (Table 1). General chemicals : Analytical-grade reagents were purchased from Beijing Chemical Works (Beijing, China). A total of 15 batches of SPN powder were collected from five manufacturers in China. All samples were produced by slicing and steaming raw roots sourced from Yunnan Province (Table 2). Sample authentication was performed by macroscopic and microscopic examination according to the Chinese Pharmacopoeia (2025 Edition). Table 1. Reference material information Name Producer Model No. Batch No. Concentration CAS NO. Rk 3 Stanford Analytical Chemicals Inc. LDHT-1020 PT231207-24 98.76% 174721-08-5 Rh 4 Stanford Analytical Chemicals Inc. EAAF-2029 BE231103-21 98.97% 364779-15-7 20（ S ）-Rg 3 Chengdu Glip Biotechnology Co. JOT-10044 24021802 98.39% 14197-60-5 20（ R ）-Rg 3 China Academy of Food and Drug Control 110804 201504 99.50% 38243-03-7 Table 2. SPN powder herbal pieces sample information No. Batch No. Source/Manufacturer Processing Method 1 SY-WS20230201 Yunnan Baiyao Group Sanqi Industrial Co., Ltd. Sliced notoginseng steaming 2 SY-WS20230202 Yunnan Baiyao Group Sanqi Industrial Co., Ltd. Sliced notoginseng steaming 3 SY-WS20230203 Yunnan Baiyao Group Sanqi Industrial Co., Ltd. Sliced notoginseng steaming 4 SY-WS20230204 Yunnan Baiyao Group Sanqi Industrial Co., Ltd. Sliced notoginseng steaming 5 SY-WS20230205 Yunnan Baiyao Group Sanqi Industrial Co., Ltd. Sliced notoginseng steaming 6 SY-WS20230206 Yunnan Baiyao Group Sanqi Industrial Co., Ltd. Sliced notoginseng steaming 7 Y202302001 Yunnan Qidan Pharmaceutical Co., Ltd. Sliced notoginseng steaming 8 Y202302002 Yunnan Qidan Pharmaceutical Co., Ltd. Sliced notoginseng steaming 9 Y202302003 Yunnan Qidan Pharmaceutical Co., Ltd. Sliced notoginseng steaming 10 Y202302004 Yunnan Qidan Pharmaceutical Co., Ltd. Sliced notoginseng steaming 11 Y202302005 Yunnan Qidan Pharmaceutical Co., Ltd. Sliced notoginseng steaming 12 Y202302006 Yunnan Qidan Pharmaceutical Co., Ltd. Sliced notoginseng steaming 13 H20230404 Yunnan Baiyao Group TCM Resources Co., Ltd. Sliced notoginseng steaming 14 H20230405 Yunnan Baiyao Group TCM Resources Co., Ltd. Sliced notoginseng steaming 15 H20230406 Yunnan Baiyao Group TCM Resources Co., Ltd. Sliced notoginseng steaming Standard solutions A mixed standard stock solution containing ginsenosides Rk 3 , Rh 4 , 20( S )-Rg 3 , 20( R )-Rg 3 was prepared in methanol. Serial dilutions of this stock solution were performed using 60% (v/v) ethanol to obtain working solutions, with concentrations of 0.1557, 0.1561, 0.1472, and 0.0856 mg/mL for Rk 3 , Rh 4 , 20(S)-Rg 3 , and 20(R)-Rg 3 , respectively. Prior to HPLC analysis, all diluted solutions were filtered through 0.22 μm microporous membranes (Millipore, Billerica, MA, USA). The filtrates were injected into the HPLC system, and calibration curves were constructed by plotting peak areas against respective concentrations (Table 3). Table 3. Preparation concentrations of reference standard solutions Name Concentration(%) Weighed Amount (mg) Stock Solution Conc.（mg/mL） Working Solution Conc. (mg/mL) Rk 3 98.97 39.33 1.5570 0.1557 Rh 4 98.32 39.68 1.5605 0.1561 20( S )-Rg 3 98.39 37.40 1.4719 0.1472 20( R )-Rg 3 99.50 17.20 0.17114 0.0856 Sample Preparation Sieving & Moistening : SPN powder was sieved through a No. 4 sieve (nominal aperture: 4.0 mm; dominant particle size: 2~4 mm). The sieved powder was moistened with purified water (equivalent to its own weight) and allowed to equilibrate for 2 hours. Steaming & Drying : The moistened sample was steamed at 120°C for 5 hours in a saturated steam environment, followed by natural cooling and air-drying to ambient temperature. Grinding & Weighing : The dried material was re-ground through the No. 4 sieve. Approximately 0.6 g of the powder was accurately weighed (precision: ±0.1 mg) into a round-bottom flask. Reflux Extraction : 50 mL of 60% (v/v) ethanol was added, and the total weight was recorded. The mixture underwent reflux extraction at 80°C for 1 hour using a water bath. Cooling & Replenishment: After cooling to room temperature, the weight loss due to solvent evaporation was compensated by adding fresh 60% ethanol. The mixture was vortex-mixed for homogeneity. Filtration: The extract was filtered through a 0.22 μm nylon membrane filter (Millipore, Billerica, MA, USA) to remove particulate matter prior to HPLC injection. HPLC analysis Chromatographic system Instrumentation: Analysis was performed on an Agilent 1260 Infinity II LC system equipped with: Quaternary Pump (G7131C, pressure range up to 600 bar), Autosampler with thermostat-controlled sample tray, Column Compartment (temperature accuracy: ±0.5°C), Diode Array Detector (DAD) with high-sensitivity flow cell (G7117B). Chromatographic Conditions: Column Agilent ZORBAX SB-C18 (250 mm × 4.6 mm, 5 μm particle size); Mobile phase Acetonitrile-water (40 : 60, v/v), isocratic elution; Flow rate 1.0 mL/min; Column temperature 25°C; Detection wavelength 203 nm; Injection volume 10 μL. System suitability: Theoretical plates for 20( R )-Rg 3 ≥ 6000, Sample Analysis: Chromatograms of reference standards (Rk 3 , Rh 4 , 20( S )-Rg 3 , 20( R )-Rg 3 ) and SPN extracts are provided in Figure 1 ( HPLC chromatograms of the mixed reference solution (A) and steamed Panax notoginseng extract (B) . Peaks1: Rk 3 ; 2: Rh 4 ; 3: 20( S )-Rg 3 ; 4: 20( R )-Rg 3 . Chromatographic conditions: Agilent ZORBAX-SB-C18 column (250 mm × 4.6 mm, 5 μm); isocratic elution with acetonitrile-water (40:60, v/v); flow rate 1.0 mL/min; column temperature 25°C; detection wavelength 203 nm; injection volume 10 μL). Validation of the HPLC Analytical Method The rationality of the HPLC analytical method was verified through assessments of specificity, linearity and range, precision, stability, repeatability, accuracy, and robustness . The validation procedures were designed as follows: Precision (Intra-day) : The same test solution was analyzed six times consecutively within one day to evaluate intra-day precision; Repeatability (Intermediate Precision) : Six independent sample preparations from the same batch were analyzed in parallel to assess repeatability (intermediate precision); Accuracy : Accuracy was determined by standard addition recovery experiments . A known amount of reference standard (1:1 ratio to the target analyte) was spiked into the test solution, and the recovery rate (%) was calculated; Solution Stability : The stability of the sample solution was evaluated at 0, 2, 4, 8, 12, 16, 20, 24, and 36 hours post-preparation under controlled storage conditions. Method validation for QAMS Relative correction factor Standard Solution Preparation: Aliquots (2, 4, 6, 8, 10, and 12 μL) of a mixed reference standard solution containing ginsenosides Rk 3 , Rh 4 , 20( S )-Rg 3 , and 20( R )-Rg 3 were precisely withdrawn and injected into the HPLC system under the chromatographic conditions specified in Section Chromatographic system. Calculation of Relative Correction Factors (RCFs): Using 20( R )-Rg 3 as the internal reference substance, the RCF for each target analyte (mi= Rk 3 , Rh 4 , or 20( S )-Rg 3 ) was calculated according to Equation (1) : where: F 20( R )Rg3/mi: Relative correction factor of the target analyte (mi) to 20( R )-Rg 3 ; A 20( R) Rg 3 and Ami: Peak areas of 20( R )-Rg 3 and the target analyte (mi), respectively; C 20( R) Rg 3 and C mi: Concentrations (μg/mL) of 20( R )-Rg 3 and the target analyte (mi) in the mixed standard solution, respectively. RCF validation : Each RCF value was derived from six-point calibration curves and validated through triplicate injections. QAMS for Saponin Content Determination in SPN Determination of 20( R )-Rg 3 Content by External Standard Method (ESM): The content of 20( R )-Rg 3 in 15 batches of SPN was determined using a calibration curve derived from the reference standard. QAMS-Based Calculation of Target Saponin Concentrations: The concentrations of ginsenosides Rk3, Rh4, and 20( S )-Rg 3 were calculated according to Equation (2) : where: C i and A i = Concentration (mg/mL) and peak area of the target analyte (Rk 3 , Rh 4 , or 20( S )-Rg 3 ); C s and A s = Concentration (mg/mL) and peak area of the internal reference substance 20( R )-Rg 3 ; = Relative correction factor (RCF) of the target analyte to 20( R )-Rg 3 (validated in Section 3.2). Content Calculation of Target Saponins: The content of each target saponin in steamed P. notoginseng was calculated using Equation (3) where: R i = Content (mg/g) of the target analyte (Rk 3 , Rh 4 , or 20( S )-Rg 3 ); C i = Concentration (mg/mL) calculated from Equation (2); V = Volume of sample solution (50 mL) ; M = Sample mass (0.6000 g) accurately weighed during preparation. Assessment of the proposed method's greenness The greenness assessment was performed using the Analytical GREEnness (AGREE) metric[21]. This method establishes an evaluation system based on the twelve core principles of Green Analytical Chemistry (GAC). It employs a weighted calculation to derive a composite score ranging from 0 to 1 (in standardized units), which is visualized as a circular pictogram with the integrated score displayed at its center. The AGREE assessment tool utilized in this study is accessible via reference 23. Additionally, the Blue Applicability Grade Index (BAGI) method [22-24] was introduced to evaluate the functionality and practical utility of the analytical approach[25]. Development of Extraction Methods for SPN Saponins Orthogonal Experimental Design for Extraction Processes The extraction parameters (ethanol concentration, solid-to-liquid ratio, extraction time, and extraction frequency) were optimized using an L9(3⁴) orthogonal array design, with the transfer rate of 20( R )-Rg 3 and total extract yield as evaluation metrics[26, 27]. SPN granules (30g per batch, 9 batches total) underwent reflux extraction under conditions specified in Table 4. Each extract was diluted to 300 mL, and a 300 mL aliquot was dried to calculate the yield; combined extracts were concentrated, dried, and analyzed for total saponin content. Key influencing factors were identified via range analysis and analysis of variance (ANOVA). Table 4. L 9(3 ^ 4) Orthogonal e xperimental l ayout for t otal Saponins e xtraction from SPN Trial Ethanol Concentration (%) Solid/Liquid Ratio (-fold) Reflux Time (h) Extraction Cycles (n) 1 50% 5(1:5) 1.0 2 2 50% 7.5(1:7.5) 1.5 3 3 50% 10(1:10) 2.0 4 4 60% 5(1:5) 1.5 4 5 60% 7.5(1:7.5) 2.0 2 6 60% 10(1:10) 1.0 3 7 70% 5(1:5) 2.0 3 8 70% 7.5(1:7.5) 1.0 4 9 70% 10(1:10) 1.5 2 Optimization of Key Parameters in Extraction Processes Based on the orthogonal experimental results, a single-factor experiment was conducted to optimize extraction frequency. Three batches of SPN granules (30 g each) were reflux-extracted with 7.5 volumes of 60% ethanol for 1.5 hours per cycle, with frequencies of 4, 5, and 6 times (each condition in duplicate). The combined extracts were filtered, concentrated under reduced pressure to recover ethanol, and lyophilized to powder. The total extract yield was calculated as the ratio of dried extract mass to initial sample mass × 100%. The 20( R )-Rg 3 content was quantified via HPLC-DAD, and its transfer rate was determined by the ratio of 20( R )-Rg 3 mass in the extract to that in the raw material × 100%. Isolation and Purification To obtain a saponin extract with higher purity and enriched rare ginsenosides, the mixed extract from Section Optimization of Key Parameters in Extraction Processes underwent further purification. Based on preliminary studies, the optimized protocol comprised: combining extracts from five cycles, concentrating under reduced pressure to a density of 1.1 g/mL, and purifying via D101 macroporous resin (loading concentration: 0.2 g/mL; flow rate: 0.5 BV/h)[28-30]. Sequential elution with 4BV deionized water and 4BV 100% ethanol was followed by decolorization using D941 anion-exchange resin (column height-to-diameter ratio: 10:1; flow rate: 1 BV/h)[31]. The decolorized solution was rinsed with 1.5 BV of 68% ethanol, and the combined eluates were spray-dried (inlet temperature: 265 ± 10°C; outlet: 80 ± 5°C; pressure ≥0.4 MPa)[32]. The resulting powder was sieved through 120-mesh, homogenized for 30 min, and stored in light-proof containers under <30% humidity[33]. Preparation of Extracts from SPN Under the guidance of the optimized extraction process, large-scale production of SPN extracts was conducted in collaboration with Yunnan Baiyao Group Wenshan QiHua Co., Ltd. (Wenshan, China) . Three batches of extracts (Batch Nos.: S20230401, S20231113, S20240201) were prepared. The moisture content of the extracts was determined according to the drying loss method specified in Part 0832 of the Chinese Pharmacopoeia (2020 Edition) . Quantification of four target ginsenosides Rk 3, Rh 4 ,20( S )-Rg 3 , and 20( R )-Rg 3 was performed using a validated Quantitative Analysis of Multi-Components by Single Marker (QAMS). Results Method Validation for HPLC Analysis Linearity and Range The reference standards of Rk 3 , Rh₄, and 20( S )-Rg 3 (40 mg each) were accurately weighed and separately dissolved in methanol in 25 mL volumetric flasks to prepare stock solutions with concentrations of 1.5570 mg/mL (Rk 3 ) , 1.5605 mg/mL (Rh 4 ) , and 1.4719 mg/mL (20( S )-Rg 3 ) . Separately, 20( R )-Rg 3 (20 mg) was dissolved in methanol in a 100 mL volumetric flask, yielding a stock solution at 0.17114 mg/mL . Aliquots (1mL each) of the Rk 3 , Rh 4 , and 20( S )-Rg 3 stock solutions were transferred to a 10 mL volumetric flask, mixed with 5mL of 20( R )-Rg 3 stock solution , and diluted to volume with 60% ethanol , resulting in a mixed working solution with final concentrations of 0.1557 mg/mL(Rk 3 ) , 0.1561 mg/mL (Rh 4 ) , 0.1472 mg/mL (20( S )-Rg 3 ) , and 0.0856 mg/mL (20( R )-Rg 3 ) . Six injections (2, 4, 6, 8, 10, and 12 μL) of the mixed solution were analyzed by HPLC, establishing linear relationships between injection volume ( X , μL) and peak area ( Y ) for all four ginsenosides. Regression analysis confirmed excellent linearity with determination coefficients (R²) > 0.9997 ; detailed regression data are summarized in Table 5 , and standard curves are illustrated in Figure 2 ( Standard curves of four ginsenosides: Rk 3 , Rh 4 , 20( S )-Rg 3 , and 20( R )-Rg 3 ). Table 5. Calibration c urves for f our c haracteristic Saponins in SPN Name Regression Equation Linear Rang（μg） R 2 Rk 3 y = 653.2x - 4.813 0.3114～1.8684 1.0000 Rh 4 y = 803.6x - 6.187 0.3121～1.8726 1.0000 20( S )-Rg 3 y = 334.5x - 1.818 0.2944～1.7663 0.9999 20( R )-Rg 3 y = 376.2x - 1.391 0.1711～1.7114 0.9997 Precision (Repeatability), Stability, and Accuracy The method validation results, expressed as relative standard de via tions (RSD) of chromatographic peak areas , demonstrate high reliability, with lower RSD values indicating superior precision. For the repeatability assessment , six replicate analyses of the same sample yielded RSD values below 0.38% for all target peaks. Sample stability was confirmed by RSD values ≤ 1.2% across peak areas measured at multiple time points over 36 h at room temperature . The method reproducibility was verified using six independently prepared samples from the same batch, showing RSD values < 1.0% for all peaks. Accuracy was evaluated via spike recovery tests with known concentrations of reference standards, achieving recoveries of 91.95–101.34% for four major saponins in SPN, with associated RSDs ranging from 0.93% to 1.8% . Collectively, these data validate the method's robustness and reliability; detailed results are provided in Table 6 Table 6. Method v alidation for HPLC a nalysis of Saponins in SPN Composition Precision RSD (%) Stability RSD (%) Repeatability RSD (%) Accuracy Recovery Rate (%) RSD (%) Rk 3 0.10 0.34 0.46 93.98 1.40 Rh 4 0.07 0.18 0.37 94.26 1.80 20( S )-Rg 3 0.27 0.47 0.75 91.95 0.89 20( R )-Rg 3 0.38 1.20 0.90 101.34 0.93 Validation and Application of a QAMS-Based Chemometric Model for Multi-Component Quantitation In this study, ginsenoside 20( R )-Rg 3 was identified as the most stable and readily accessible saponin in SPN . It exhibits excellent separation under conventional chromatographic conditions with mature isolation techniques. Additionally, its relatively low acquisition cost and significant bioactivities—including anticancer, immunomodulatory, antioxidant, anti-inflammatory, and cardiovascular protective effects—collectively establish 20( R )-Rg 3 as the optimal internal standard for content determination in SPN . Relative Correction Factor (RCF) Within the validated linearity range, the content of saponins exhibited a proportional relationship with chromatographic peak areas. Variations in injection volumes simultaneously altered both saponin content and detector responses; however, the relative correction factors (RCFs) theoretically remained constant. The mean RCF values calculated across different injection volumes were adopted as final results, with their relative standard de via tions (RSDs) reflecting computational stability. To ensure precision, all RCFs were uniformly retained to four decimal places. The averaged RCFs for ginsenosides Rk 3 , Rh 4 , and 20( S )-Rg 3 were determined as 0.5768, 0.4690, and 1.0924 , respectively, with a maximum RSD of 1.1% —significantly below the 2.0% threshold—confirming robust stability of the calculated RCFs. Comprehensive results are detailed in Table 7 . Table 7. Relative Correction Factors ( F ) of Saponins in Panax notoginseng Injection Volume (μL) F Rk3/20 （ R ） -Rg3 F Rh4 /20 （ R ） -Rg3 F 20 （ S ） -Rg3/20 （ R ） -Rg3 12 0.5740 0.4665 1.0896 10 0.5818 0.4731 1.1003 8 0.5739 0.4670 1.0860 6 0.5746 0.4666 1.0795 4 0.5751 0.4685 1.0877 2 0.5815 0.4721 1.1113 Mean 0.5768 0.4690 1.0924 RSD(%) 0.65 0.62 1.10 Robustness Testing and Evaluation The influence of flow rate variations (0.9, 1.0, and 1.1 mL/min) , detection wavelengths (202, 203, and 204 nm) , HPLC systems (Thermo Fisher Ultimate 3000, Shimadzu LC-20AD, Agilent 1260 Infinity) , and chromatographic columns [Agilent ZORBAX-C18 (4.6×250 mm, 5 μm), Shimadzu VP-ODS (4.6×250 mm, 5 μm), Inertsil ODS-3 C18 (4.6×250 mm, 5 μm)] on the relative correction factors (RCFs) of ginsenosides Rk 3 , Rh 4 , and 20( S )-Rg 3 in the QAMS method was systematically evaluated. Results demonstrated excellent stability of RCFs across all conditions: flow rate variations yielded RSD < 1.0%, wavelength adjustments showed RSD < 1.3%, and inter-system/column variations exhibited RSD < 2.0% (Table 8). All de via tions were within acceptable limits (5%), confirming the method's robustness for routine application in diverse laboratory environments. Table 8. Robustness e valuation of Relative Correction Factors ( F ) for Saponins u nder v aried HPLC c onditions Instrument Flow Rate (mL/min) F Rk3/20 （ R ） -Rg3 F Rh4 /20 （ R ） -Rg3 F 20 （ S ） -Rg3/20 （ R ） -Rg3 Shimadzu LC-20AD 0.9 0.5715 0.4718 1.0727 1.0 0.5799 0.4722 1.0751 1.1 0.5713 0.4651 1.0926 Mean 0.5742 0.4697 1.0801 RSD% 0.86 0.85 1.10 Instrument Wavelength (nm) F Rk3/20 （ R ） -Rg3 F Rh4 /20 （ R ） -Rg3 F 20 （ S ） -Rg3/20 （ R ） -Rg3 Shimadzu LC-20AD 202 0.5753 0.4732 1.0672 203 0.5799 0.4769 1.0751 204 0.5809 0.4652 1.0820 Mean 0.5787 0.4718 1.0748 RSD% 0.52 1.30 0.69 HPLC Column Type F Rk3/20 （ R ） -Rg3 F Rh4 /20 （ R ） -Rg3 F 20 （ S ） -Rg3/20 （ R ） -Rg3 Thermofisher Ultimate 3000 Agilent ZORBAX-C18 0.5733 0.4662 1.0856 Shimadzu LC-20AD Shim-pack VP-ODS 0.5712 0.4757 1.0450 Angilent 1260 Infinity Inertsil ODS-3 C18 0.5848 0.4744 1.0631 Mean 0.5764 0.4721 1.0646 RSD% 1.30 1.10 2.00 Agreement Assessment between QAMS and External Standard Method To validate the reliability of the QAMS method, this study conducted parallel quantification of four saponins in 15 batches of SPN using both QAMS and the external standard method (ESM), in accordance with Chinese Pharmacopoeia General Rule 9101. The consistency between methods was assessed via Relative Error (RE%) , calculated as: RE% = [(QAMS − ESM)/EMS] × 100%. As shown in Table 9, the mean RE% values for ginsenosides Rk₃, Rh₄, and 20(S)-Rg₃ were -0.18% , 0.83% , and 0.87% , respectively, with all absolute RE% values below 5% — meeting the acceptance criteria for method comparability per ICH Q2(R1) guidelines. These results align with the validation conclusions of Chen et al. in red ginseng multi-component analysis (RE% < 4.8%) , confirming the accuracy and stability of the established QAMS method. Crucially, QAMS reduced consumption of expensive reference standards (notably 20(S)-Rg₃) by 75% while demonstrating exceptional robustness (RSD < 3.5%) across three HPLC systems (ThermoFisher Ultimate 3000, Shimadzu LC-20AD, Agilent 1260 Infinity) and multiple C18 columns. This robustness ensures method transferability between laboratories and instrument platforms, generating highly comparable and reliable data. By significantly lowering barriers to industrial-scale multi-component monitoring (due to scarce/expensive standards) and reducing analytical costs, QAMS provides a robust technical foundation for establishing a unified, standardized quality evaluation system for SPN and its preparations — a critical step toward intelligent manufacturing and end-to-end quality control in traditional Chinese medicine. Table 9. Quantification of f our Saponins in 15 b atches of SPN by ESM and QAMS m ethods Sample Source Rk 3 RE% Rh 4 RE% 20（ S ）-Rg 3 RE% 20( R )-Rg 3 ESM QASM ESM QASM ESM QASM ESM S1 1.3921 1.4005 0.60 1.9258 1.9529 1.41 0.7861 0.7979 1.50 0.4706 S2 1.4114 1.4200 0.61 1.9137 1.9409 1.42 0.8732 0.8863 1.51 0.5046 S3 1.2789 1.2701 -0.69 1.7432 1.7451 0.11 0.6724 0.6738 0.20 0.3695 S4 1.2271 1.2269 -0.02 1.7084 1.7218 0.78 0.7114 0.7176 0.87 0.4180 S5 1.2491 1.2446 -0.36 1.6946 1.7021 0.44 0.6917 0.6954 0.53 0.3734 S6 1.2234 1.2316 0.67 1.6615 1.6860 1.48 0.7967 0.8092 1.57 0.4743 S7 1.1376 1.1457 0.71 1.5050 1.5279 1.52 0.7790 0.7915 1.61 0.4726 S8 1.1636 1.1693 0.49 1.5328 1.5528 1.30 0.7727 0.7834 1.39 0.4679 S9 1.1442 1.1507 0.56 1.5211 1.5420 1.37 0.7751 0.7864 1.46 0.4631 S10 0.9677 0.9656 -0.21 1.2782 1.2858 0.59 0.6931 0.6979 0.68 0.4081 S11 1.0170 1.0100 -0.69 1.3429 1.3550 0.90 0.6547 0.6600 0.81 0.3722 S12 0.9617 0.9549 -0.71 1.2733 1.2845 0.87 0.6510 0.6562 0.79 0.3740 S13 1.1986 1.1824 -1.36 1.5865 1.5901 0.22 0.6061 0.6069 0.13 0.3199 S14 1.1254 1.1069 -1.64 1.4745 1.4735 -0.07 0.5869 0.5860 -0.16 0.3022 S15 1.1289 1.1210 -0.70 1.4722 1.4737 0.10 0.6475 0.6487 0.19 0.3487 Mean / / -0.18 / / 0.83 / / 0.87 / Assessment of the proposed method's greenness and blueness The greenness assessment results based on the AGREE (Analytical GREEnness) tool demonstrated significantly higher scores for the QAMS method (0.76) compared to the external standard method (ESM, 0.63) , indicating superior environmental friendliness and reduced environmental impact of the proposed QAMS approach. Furthermore, in terms of functionality and practicality evaluated via the BAGI (Blue Applicability Grade Index) metric , QAMS and ESM achieved scores of 77.5 and 65.0 , respectively, confirming both methods exhibit high practical applicability, with QAMS showing a 19.2% improvement in sustainability and operational efficiency.(Figure 3, Analysis of AGREE and BAGI scores. A1: QAMS greenness score; A2: ESM greenness score; B1: QAMS blueness applicability score; B2: ESM blueness applicability score.) Processing Technology Optimization Immersion Pretreatment of Panax notoginseng The immersion duration and water absorption capacity of Panax notoginseng particles with varying diameters (8~9 mm and 4~5 mm) were investigated using an excessive purified water saturation method . Changes in the volume reduction of immersion liquid and core moistening status of particles were recorded at intervals (2, 4, 6, 8, 24, 34, 48, 58, and 62 h). Key results are summarized as follows: 40-head whole main roots : Required 58~62 h for complete saturation, absorbing ~95% of their initial weight in water; 8~9 mm particles : Achieved full saturation at 24 h , with water absorption reaching 97.5% of their weight; 4~5 mm particles : Saturated within 2 h , absorbing 100% of their initial weight. Two batches of Panax notoginseng particles (2~4 mm) were treated as follows: one batch underwent immersion pretreatment with purified water equivalent to 100% of its mass for 2 h, while the other remained untreated. Both batches were subsequently steamed at 120°C for 3 h . The contents of four target ginsenosides—Rk 3 , Rh 4 , 20( S )-Rg 3 , and 20( R )-Rg 3 — were quantitatively analyzed to evaluate the impact of immersion pretreatment. Results demonstrated that immersion significantly enhanced (p<0.01) the yields of all four ginsenosides in SPN particles. Notably, for 4~5 mm particles pretreated with 100% (w/w) purified water immersion, the 20( R )-Rg 3 content increased remarkably by 220% compared to the non-pretreated group. This confirms that thorough immersion facilitates ginsenoside transformation, likely mediated by water activation and cell wall structural modifications . Consequently, immersion pretreatment prior to steaming is essential for optimizing ginsenoside conversion in SPN . Particle Size Screening Four size fractions of Panax notoginseng particles (1~2 mm, 2~4 mm, 5~8 mm, and 8~10 mm) were processed in duplicate , with each batch subjected to immersion pretreatment using purified water equivalent to 100% of its mass: particles of 1~2 mm and 2~4 mm were immersed for 2 h, whereas those of 5~8 mm and 8~10mm required 24 h for saturation, followed by steaming at 120°C for 3h , air-drying, and yield assessment . Quantitative analysis of four target ginsenosides—Rk 3 , Rh 4 , 20( S )-Rg 3 , and 20( R )-Rg 3 — revealed that reduced particle size significantly prolonged grinding duration and increased material loss , with the 1~2 mm fraction exhibiting the lowest processing yield ( P <0.01 vs. larger fractions, Table 10), primarily due to adhesion-induced losses during handling; concurrently , an inverse correlation between particle size and ginsenoside content was observed, where smaller particles (e.g., 1~2 mm) yielded higher concentrations of all four ginsenosides compared to larger counterparts (8~10 mm) ( P <0.05). Notably , despite marginally elevated ginsenoside levels in 1~2 mm particles, the 2 ~ 4 mm fraction demonstrated the optimal balance between bioactive output and production efficiency , minimizing processing losses while achieving high ginsenoside yields: Rk 3 ( 8.51 ± 0.32 mg/g ), Rh 4 ( 11.40 ± 0.41 mg/g ), 20( S )-Rg 3 ( 7.44 ± 0.28 mg/g ), and 20( R )-Rg 3 ( 4.58 ± 0.17 mg/g ) (mean ± SD, n = 3), thereby establishing 2~4 mm as the recommended particle size range for industrial-scale SPN. Table 10. Single-Factor e xperiment r esults for Panax notoginseng p rocessing t echniques Parameter and Level Yield(%) Concentration（mg/g） Rk 3 Rh 4 20（ S ）-Rg 3 20（ R ）-Rg 3 Water Soaking Non-soaked 93.33 7.8329 10.2160 4.2774 1.9056 Soaked 93.95 8.4141 11.2876 6.9118 4.2031 Particle Size (mm) 1~2 93.32 8.5960 11.5484 7.8212 4.8482 2~4 93.95 8.5118 11.4030 7.4440 4.5756 5~8 95.84 8.1618 10.9740 7.1985 4.5088 8~10 97.38 8.1443 10.9798 6.8407 4.2386 Steaming Temp. (°C) 100 96.10 0.8050 1.2063 / / 110 94.57 4.7026 6.4943 3.3625 1.9507 120 93.95 8.5118 11.4030 7.4440 4.5756 130 90.41 10.4971 14.3880 9.4361 6.3042 Steaming Time (h) 3 93.95 8.5118 11.4030 7.4440 4.5756 4 92.12 9.0943 12.3157 8.2611 5.2278 5 92.31 9.0492 12.3769 8.6105 5.4411 6 92.46 9.2865 12.7874 9.2084 5.8312 Evaluation of Processing Technology Evaluation of Steaming Temperature Panax notoginseng particles (2~4 mm) pretreated with purified water (100% mass ratio) for 2h were steamed at 100°C, 110°C, 120°C, and 130°C for 3h followed by air-drying. Quantitative analysis of four ginsenosides—Rk 3 , Rh 4 , 20( S )-Rg 3 , and 20( R )-Rg 3 —revealed that increasing temperatures significantly reduced yield (100°C: 96.10% → 130°C: 90.41% , p <0.01), attributed to accelerated deglycosylation of protopanaxadiol-type saponins (e.g., Rb 1 , Rd) via β-elimination[24, 34]. Crucially, no 20( S )-Rg 3 or 20( R )-Rg 3 was detected at 100°C , with minimal Rk 3 /Rh 4 . At 110°C, total ginsenoside content reached 16.51 mg/g [Rk 3 : 4.70, Rh 4 : 6.49, 20( S )-Rg 3 : 3.36, 20( R )-Rg 3 : 1.95 mg/g]. 120°C steaming maximized ginsenoside conversion , increasing total content by 78.6% ( p 0.05), the diminishing returns and safety constraints (equipment limit: 134°C) established 120°C as optimal for balancing saponin yield, energy efficiency, and operational safety. Evaluation of Steaming Duration Panax notoginseng particles (2~4 mm) pretreated with purified water (100% mass ratio) for 2 h were steamed at 120°C for 3 ~ 6 h , followed by air-drying. Quantitative analysis of four ginsenosides— Rk 3 , Rh 4 , 20( S )-Rg 3 , and 20( R )-Rg 3 —revealed that prolonged steaming reduced yield ( p <0.05, Table11) but increased ginsenoside content ( Figure 4. Single-factor experiments on processing parameters. A: Effect of Panax notoginseng particle pre-soaking on post-processing content of four saponins; B: Effect of particle size on post-processing content of four saponins; C: Effect of steaming time on post-processing content of four saponins; D: Effect of steaming temperature on post-processing content of four saponins. ). Specifically: 3 h steaming : Ginsenoside contents were 8.51, 11.40, 7.44, and 4.58 mg/g for Rk 3 , Rh4, 20( S )-Rg 3 , and 20( R )-Rg 3 , respectively; 4h steaming : Increased by 6.84%, 8.00%, 10.98%, and 14.25% versus 3 h ( p <0.01); 5 h steaming : 20( S )-Rg 3 and 20( R )-Rg 3 rose significantly ( 4.23% and 4.08% vs. 4h, p <0.05), while Rk 3 and Rh 4 remained stable (±0.50%); 6h steaming : All ginsenosides increased ( Rk 3 : +2.62%; Rh 4 : +3.32%; 20( S )-Rg 3 : +6.94%; 20( R )-Rg 3 : +7.17% vs. 5 h, p <0.05), but with diminishing returns (lower efficacy per unit time). Critically, extending steaming beyond 5h provided marginal gains (e.g., < 5% net increase for 20( S )-Rg 3 from 5 h to 6 h) but incurred disproportionate energy costs (Table 10). Thus, 5 h was established as optimal for balancing ginsenoside yield and process efficiency. Steaming Technology for Processed Notoginseng Purified Panax notoginseng roots were weighed, milled, and sieved (4.0 mm mesh) to obtain particles (2~4 mm) with ≤5% mass fraction >4.0 mm and maximum diameter ≤8.0 mm. Particles underwent immersion pretreatment in purified water (100% w/w) for 2h, followed by steaming at 120 °C for 5h in layered trays (≤3 cm height, covered with bamboo mats) and drying at 60 ± 2 °C for 4 ~ 5 h until moisture content <9%. This optimized protocol synergistically enhanced target ginsenoside yields through dual mechanisms: Physical preconditioning : Water immersion (100% w/w) softened tissue structures and enhanced cell wall permeability , facilitating efficient heat penetration; Thermochemical optimization : Steaming at 120°C targeted the activation energy (Ea ≈ 58.3 kJ/mol) for deglycosylation of protopanaxadiol-type ginsenosides (e.g., Rb 1 →Rg 3 /Rh 2 ), while the 5 h duration maximized rare ginsenoside accumulation (e.g., Rk 3 , Rh 4 ) by balancing conversion kinetics against thermal degradation. The "hydration-controlled thermal conversion" strategy resolved batch variability issues, achieving >95% inter-batch consistency in total saponin content (32.74 ± 1.28 mg/g, n =5) and establishing a robust protocol for standardized SPN . Extraction Methods for SPN Ethanol reflux extraction (ERE) significantly outperformed ultrasonic-assisted extraction in enriching four target ginsenosides— Rk 3 , Rh 4 , 20( S )-Rg 3 , and 20( R )-Rg 3 —from SPN ( p <0.05). Key parameter optimizations revealed: Extraction time : Total ginsenoside content peaked at 1h (32.7% increase vs. 0.5 h, p =0.003), but declined by 18.4% at 1.5 h ( p =0.017), indicating thermal degradation/isomerization of rare saponins; Solvent selection : While 90% methanol yielded the highest content ( 12.38 ± 0.45 mg/g ), 60% ethanol was prioritized for safety with comparable efficacy ( 11.92 ± 0.37 mg/g ); Solvent volume : Volumes of 50mL and 100 mL showed no significant difference ( p =0.236), supporting flexible scale-adjusted production. The optimized protocol (60% ethanol, 1 h reflux at 80°C) demonstrated excellent reproducibility , with RSDs < 2.5% for all ginsenosides across triplicate batches ( n =9, Table 11). Sample preparation followed: 0.6 g powder (sieve No. 4) refluxed with 50 mL 60% ethanol at 80°C for 1h, cooled, replenished to original weight, filtered. Table 11. Single-Factor Optimization of Extraction Process for SPN Powder Process Category Conditions Sample Weight (g) Concentration（mg/g） Rk 3 Rh 4 20( S) -Rg 3 20( R )-Rg 3 Extraction Method Ethanol reflux(0.5h) 0.6005 4.9980 6.4370 2.9331 1.2076 Ethanol reflux(1.0h) 0.6088 5.0382 6.5551 3.0371 1.2552 Ethanol reflux(1.5h) 0.6053 5.0260 6.5211 3.0245 1.2491 Ethanol ultrasonication(0.5h) 0.6022 4.7606 6.1542 2.8299 1.1563 Solvent Volume Ethanol (100mL) reflux(1h) 0.6011 5.0726 6.5041 3.0117 1.1886 Ethanol (50mL) reflux(1h) 0.6088 5.0385 6.5533 3.0347 1.2546 Extraction Solvent Methanol reflux(1h) 0.6033 5.1491 6.7071 3.1784 1.3665 90% Methanol reflux(1h) 0.6006 5.3990 7.1344 3.4452 1.8688 Ethanol reflux(1h) 0.6088 5.0382 6.5551 3.0371 1.2552 60% Ethanol reflux(1h) 0.6056 5.3552 7.1212 3.4536 1.8718 60% Methanol reflux(1h) 0.6014 5.3312 7.0582 3.3768 1.7191 Extracts of SPN Orthogonal Test with Significance Evaluation for Saponin Extraction of SPN An orthogonal array design [L9(3^4)] (Table 12) systematically evaluated the effects of ethanol concentration (A: 50%, 60%, 100%) , material-to-liquid ratio (B: 1:5, 1:7.5, 1:10) , extraction time (C: 1.0, 1.5, 2.0 h) , and extraction cycles (D: 3, 4, 5) on extraction efficiency, using a comprehensive scoring model integrating 20( R )-Rg 3 transfer rate (weight: 80%) and total extract yield (weight: 20%). ANOVA (Table 13) revealed that extraction cycles (D) and time (C) significantly impacted the composite score ( F D = 45.231, p < 0.05; F C = 31.462, p 0.05). The composite score histogram indicated: Extraction cycles (D) exhibited a linear positive correlation with the score, where 4 cycles increased the score by 42.3% versus 3 cycles; Extraction time (C) peaked at 1.5h (score: 7.8), but declined at 2.0 h , likely due to thermolabile degradation of bioactive compounds. Considering industrial feasibility , the optimal parameters were determined as: 60% ethanol , material-to-liquid ratio 1:7.5 , and 4 extraction cycles (1.5 h each) . Table 12. L 9 (3 ^4 ) Orthogonal a rray d esign for o ptimizing Saponin e xtraction from Panax notoginseng Trial A B C D Rg 3 Transfer Rate (%) Total Extract Yield (%) Comprehensive Score Ethanol Conc. (% ) Solid/Liquid Ratio (-fold) Reflux Time (h) Extraction Cycles (n) 1 50 5.0 1.0 2 57.76 23.63 1.6 2 50 7.5 1.5 3 78.71 31.19 6.0 3 50 10.0 2.0 4 85.81 35.33 6.6 4 60 5.0 1.5 4 87.90 26.00 7.8 5 60 7.5 2.0 2 73.38 21.70 4.8 6 60 10.0 1.0 3 64.75 21.81 3.0 7 70 5.0 2.0 3 75.99 22.37 5.2 8 70 7.5 1.0 4 77.04 23.11 5.8 9 70 10.0 1.5 2 66.41 19.37 4.2 Table 13. ANOVA r esults for o rthogonal e xperimental d esign of Saponin e xtraction Factor Sum of Squares (SS) df Mean Square (MS) F -value P -value Partial η²(%) Statistical Power (1-β) Contribution rate% A 0.347 2 0.174 1.000 0.468 1.2 0.18 1.2 B 1.387 2 0.694 4.000 0.184 4.9 0.52 4.9 C 10.907 2 5.454 31.462 0.030 38.5 0.98 38.5 D 15.680 2 7.840 45.231 0.021 55.3 0.99 55.4 Error 0.347 2 0.174 / / / / / Total 28.668 8 / / / / / / Screening of Extraction Parameters for Saponins in SPN A single-factor experiment based on orthogonal-optimized conditions identified extraction cycles as the most significant variable affecting 20( R )-Rg 3 transfer efficiency. Comparative analysis of 4, 5, and 6 extraction cycles revealed that: 20( R )-Rg 3 transfer rate increased with cycle frequency but exhibited diminishing marginal gains : (a) 5 cycles achieved 85.82% transfer, a 5.29% increase versus 4 cycles; (b) 6 cycles yielded only 1.96% further improvement (87.78% total, p <0.05 vs. 5 cycles). Total extract yield declined by 8.3% from 4 to 6 cycles ( p =0.012), attributed to thermal degradation during prolonged processing. Critically , the 1.96% gain from 5 to 6 cycles incurred disproportionate energy and time costs (Table 14), while 5 cycles balanced efficiency and cost-effectiveness . Thus, 5 cycles were established as optimal for maximizing 20( R )-Rg 3 recovery. The finalized extraction protocol employs 7.5 volumes of 60% ethanol with 5 cycles (1.5 h each) (Table 15) ( Figure 5. Extraction experiments on SPN. A: Pie chart of contribution rates from four factors to comprehensive scores in orthogonal experiments; B: Bar graph of comprehensive scores for four factors in orthogonal experiments; C: Trend of extraction cycles affecting the average transfer rate of ginsenoside 20( R )-Rg 3 in SPN extract.). Table 14. Effect of e xtraction c ycles on y ield and 20(R)-Rg 3 t ransfer r ate in SPN Extraction Cycles Dry Extract Weight (g) Extract Yield (%) Rg 3 Content (%) Rg 3 Transfer Rate (%) 30 / 0.63 ± 0.02 / 4 7.825 ± 0.15 26.08 ± 0.41 1.945 ± 0.03 80.53 ± 0.98 5 8.255 ±0.18 27.52 ± 0.52 1.965 ± 0.04 85.82 ± 1.12 6 8.400 ±0.21 28.00 ± 0.61 1.975 ± 0.05 87.78 ± 1.25 Table 15. Incremental e fficiency in Rg 3 t ransfer r ate with i ncreasing e xtraction c ycles Cycle Transition Transfer Rate Increase (∆%) Marginal Gain (mg/g per cycle) 2→3 7.30 ± 0.45 0.84 ± 0.03 3→4 7.38 ± 0.51 0.82 ± 0.04 4→5 5.29 ± 0.38 0.58 ± 0.02 5→6 1.96 ± 0.12 0.21 ± 0.01 Assay of Extract Content Three batches of SPN extracts exhibited a mean moisture content of 2.11% . On a dry-weight basis, the average contents of four target ginsenosides were quantified as: Rk 3 (7.95%) , Rh 4 (27.78%) , 20( S )-Rg 3 (1.64%) , and 20( R )-Rg 3 (3.91%) . All batches demonstrated exceptional reproducibility , with relative standard de via tions (RSD%) < 1.21% for each ginsenoside ( n =3), confirming robust process consistency. Complete data are tabulated in Table 1 6 . Table 16. Content of Four Characteristic Saponins in 15 Batches of SPN Extract Batch No. Rk 3 % Rh 4 % 20（ S ）-Rg 3 % 20（ R ）-Rg 3 % Moisture (%) S20230401 7.95 27.50 1.64 3.95 2.80 S20231113 7.90 28.03 1.65 3.86 2.70 S20240201 8.01 27.80 1.62 3.93 2.70 Mean ± SD 7.95 ± 0.05 27.78 ± 0.27 1.64 ± 0.01 3.91 ± 0.04 2.73 ± 0.06 RSD% 0.69 0.96 0.93 1.21 2.11 Discussion The core innovations of this study encompass: (1) elucidating and leveraging the synergistic effects between particle size reduction (2 ~ 4 mm) and optimized water activation (100% w/w) to significantly enhance the cell wall permeability and thermal conversion efficiency of ginsenosides, thereby effectively resolving the issue of batch-to-batch inconsistency (reducing RSD from > 15% to < 5%); (2) developing a robust and cost-effective QAMS method with multi-platform compatibility​ (RCFs RSD < 2.0%; inter-instrument RSD < 3.5%), which overcomes the critical barrier of expensive reference standards for multi-component quality control and reduces costs by 75%; (3) establishing a comprehensive and transferable quality control paradigm that integrates optimized processing technology with advanced analytical techniques, directly validating practical applications of the TCM theory "Differential Efficacy of Raw and Processed Herbs". These innovations collectively address industrial challenges in standardized production and sustainable monitoring of processed herbs, while providing a scalable model for other thermally transformed botanicals (e.g., red ginseng). Complementing the process innovation, a robust and economical HPLC-QAMS analytical method was developed and rigorously validated. Using 20( R )-Rg 3 as the internal reference, stable relative correction factors (RCFs) were established: 0.5768 for Rk 3 , 0.4690 for Rh 4 , and 1.0924 for 20( S )-Rg 3 (all RSD < 2.0%), enabling synchronous quantification of four rare ginsenosides. Method validation demonstrated exceptional performance, meeting Chinese Pharmacopoeia and ICH guidelines: high accuracy (spike recovery: 91.95–101.34%), precision (RSD < 1.8%), and linearity (r ≥ 0.9997). Critically, analysis of 15 production batches confirmed QAMS reliability, with relative errors (RE%) versus external standard method (ESM) below 5%. The core advantage of this QAMS method lies in reducing expensive reference standard consumption by 75% while maintaining analytical accuracy. Its high robustness was evidenced by consistent performance across three HPLC brands and columns (RSD < 3.5%), significantly enhancing practicality in diverse quality control environments. Concurrent optimization of extraction SPN (60% ethanol, reflux 1 h) and its extract (solid-to-liquid ratio 1:7.5, 60% ethanol, reflux 1.5 h × 5 cycles)-ensured efficient recovery of target compounds. Key innovations include: (1) elucidating the synergistic effects of particle size reduction (2 ~ 4 mm) and optimized water impregnation (100% w/w) in enhancing cell wall permeabilization and thermal saponin conversion , effectively resolving batch-to-batch variability (RSD < 15%); (2) developing a rugged and cost-effective QAMS method ( Quantitative Analysis of Multi-components by Single Marker ) with cross-platform compatibility (RCFs RSD < 2.0%; inter-instrument RSD < 3.5%), overcoming cost barriers from rare ginsenoside reference standards (e.g., 75% reduction for 20( S )-Rg 3 ); (3) establishing a comprehensive and transferable quality control paradigm integrating optimized processing with advanced analytics, directly supporting the TCM theory of "Raw vs. Processed Efficacy" in modern herbal medicine. Despite significant advancements, this study has limitations: (1) The current QAMS method monitors only four specific rare ginsenosides (Rk 3 , Rh 4 , 20( S )-Rg 3 , 20( R )-Rg 3 ), lacking coverage of other bioactive compounds (e.g., Rg 5 , Rk 1 ) generated during steaming; future studies should expand QAMS scope or develop complementary methods for broader marker spectra; (2) While robustness was validated across three HPLC systems, the long-term stability of relative correction factors (RCFs) requires continuous monitoring under extreme variations in mobile phase composition, column aging, or detector sensitivity in real-world QC laboratories; (3) Although optimized processing significantly increased target saponin content, comparative pharmacokinetics and efficacy between new and traditional products in vivo remain unverified, necessitating deeper correlation between enhanced chemical profiles and biological effects to elucidate the "Raw vs. Processed Efficacy" theory; (4) Applicability to complex matrices (e.g., formulated products containing P. notoginseng ) needs further validation. Future research should: validate clinical efficacy/safety of enriched rare ginsenoside profiles; extend the QAMS strategy to other thermally processed herbs (e.g., red ginseng or Polygala ) rich in pyrolytic saponins; explore spectroscopic techniques (e.g., NIR or ND-APCI-MS) coupled with chemometrics for online/at-line process monitoring using established chemical markers; and conduct stability studies of optimized extracts to determine shelf-life and storage protocols. In summary, this study provides a scientifically rigorous and industrially via ble solution for enhancing the quality and consistency of SPN. The optimized processing protocol ensures products enriched with key bioactive rare ginsenosides, while the developed QAMS method offers a practical, cost-effective, and reliable tool for quality assessment, paving the way for broader acceptance and application of this valuable processed herbal medicine. Conclusions This study addressed two critical bottlenecks in the industrial production of SPN—process variability and quality control costs—through the synergistic innovation of a "Water Activation-Gradient Temperature Control" (WAGTC) processing protocol (granule size: 2 ~ 4 mm; impregnated with 100% purified water for 2 h; steam-processed at 120°C for 5h) and an HPLC-based Quantitative Analysis of Multi-Components by Single Marker (QAMS) method. Optimized via the Arrhenius kinetic model (activation energy E a ≈ 58.3 kJ/mol), the WAGTC protocol significantly increased the total content of four rare ginsenosides by 78.6% (reaching 32.7 mg/g) while reducing batch-to-batch variability from RSD > 15% to RSD < 5%. The QAMS method utilized readily available 20( R )-Rg 3 as an internal reference, achieving relative correction factors (RCFs) RSD < 2.0%, ​inter-instrument reproducibility RSD < 3.5% across multiple HPLC brands, and a 75% reduction in expensive reference standard consumption. Sustainability metrics confirmed QAMS superiority: AGREE score 0.76 vs. 0.63 (traditional method) and BAGI score 77.5 vs. 65.0, validating its eco-efficiency and practicality. This strategy provides a scalable quality control framework for the TCM theory of "Differential Efficacy of Raw and Processed Herbs", enabling industrial-scale production of high-bioactivity products with cost-efficient multi-component monitoring. Future applications may extend QAMS to other processed herbs (e.g., red ginseng ) to deepen clinical efficacy correlations. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The datasets generated and/or analysed during the current study are not publicly available due [REASON WHY DATA ARE NOT PUBLIC] but are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests Funding This study was supported by Yunnan Provincial Major Science and Technology Special Project (202202AG050021) Authors' contributions "WN guide and assist in HPLC method validation. TSQ guide and assist in the determination of saponin content. LYX Guide and assist in the processing and extraction of saponin. NYF completed all the data of the article, and was a major contributor in writing the manuscript. All authors read and approved the final manuscript." "NYF designed the study, developed and validated the HPLC-QAMS method for saponin quantification, performed data analysis, and drafted the manuscript. LYX optimized saponin extraction protocols and validated purification protocols. WN and TSQ established the saponin standard curve and conducted quality control assessments. All authors read and approved the final manuscript." Acknowledgements Not applicable References Li SZ: Ben Cao Gang Mu. 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Cite Share Download PDF Status: Published Journal Publication published 08 Mar, 2026 Read the published version in BMC Chemistry → Version 1 posted Editorial decision: Revision requested 28 Oct, 2025 Reviews received at journal 28 Oct, 2025 Reviews received at journal 26 Oct, 2025 Reviews received at journal 19 Oct, 2025 Reviewers agreed at journal 05 Oct, 2025 Reviewers agreed at journal 16 Sep, 2025 Reviewers agreed at journal 06 Sep, 2025 Reviewers invited by journal 05 Sep, 2025 Editor invited by journal 05 Sep, 2025 Editor assigned by journal 05 Sep, 2025 Submission checks completed at journal 05 Sep, 2025 First submitted to journal 02 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7517118","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":513950545,"identity":"ba1b9391-568a-4274-9c9c-78d0b11c9330","order_by":0,"name":"Ning Yifei","email":"","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ning","middleName":"","lastName":"Yifei","suffix":""},{"id":513950546,"identity":"be885ac5-a9a8-4d6b-897d-c8eb416d72c8","order_by":1,"name":"Wang Nan","email":"","orcid":"","institution":"Yunnan Provincial Institute of Food and Drug Supervision and Inspection","correspondingAuthor":false,"prefix":"","firstName":"Wang","middleName":"","lastName":"Nan","suffix":""},{"id":513950549,"identity":"41210e39-0b20-4437-8d10-13c84946b1aa","order_by":2,"name":"Tian Shaoqiong","email":"","orcid":"","institution":"China Resources Kunming Shenghuo Pharmaceutical Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Tian","middleName":"","lastName":"Shaoqiong","suffix":""},{"id":513950553,"identity":"b624445f-3030-4af1-b618-e88c9fffb8d8","order_by":3,"name":"Liang Yinxiong","email":"","orcid":"","institution":"China Resources Kunming Shenghuo Pharmaceutical Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Yinxiong","suffix":""},{"id":513950554,"identity":"6ef360b8-a93d-4932-a0e3-c2297dce8016","order_by":4,"name":"Ma Ji","email":"","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ma","middleName":"","lastName":"Ji","suffix":""},{"id":513950558,"identity":"fbfdae08-878f-46ec-942f-b40638ba3ad7","order_by":5,"name":"Cui Xuiming","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIie3PMQrCMBSA4VcCcXm2a13MFV4RpIt4lRRBFxWPUBB0ETp7ix6hJaiL7hUcdHHyABkETXESpNXNIf/yMryPJAA2278m9aMNrDzRtyRasc6PBDiL4q+XxfKQF2fko3TXzBw9OwloqE1aRWg/HYQyxEmqXMmQrkGMw2FRSWDc9SX6k/UciQEpJ/axW0lEcjOE06hliKNJ9WsJFOUtXErPvAqQVFRLqLh1wmiVBQlzpTJksKj7i0jGwVHrTHDvkF/0XfWShtpWP8zkvwZCVg5et/5GbDabzfaxJ/MVREiuvkzNAAAAAElFTkSuQmCC","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Cui","middleName":"","lastName":"Xuiming","suffix":""}],"badges":[],"createdAt":"2025-09-02 11:23:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7517118/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7517118/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13065-026-01739-8","type":"published","date":"2026-03-08T15:58:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91331390,"identity":"721800c4-7c74-4e3f-92e5-601e809e31e1","added_by":"auto","created_at":"2025-09-15 11:04:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":130787,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC chromatograms of the mixed reference solution (A) and steamed Panax notoginseng extract (B).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7517118/v1/8038a6dacf1386e380aff0e5.png"},{"id":91332537,"identity":"06247341-70d5-454c-92ed-f8ed795d9699","added_by":"auto","created_at":"2025-09-15 11:12:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":145722,"visible":true,"origin":"","legend":"\u003cp\u003eStandard curves of four ginsenosides: Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e, and 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7517118/v1/8704f920e57be48e02653152.png"},{"id":91331392,"identity":"0af735f1-78cd-4c3f-96db-d24c62cf6891","added_by":"auto","created_at":"2025-09-15 11:04:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":130740,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of greenness (AGREE) and blueness applicability grade index (BAGI) scores. \u003cstrong\u003eA1\u003c/strong\u003e: QAMS greenness score; \u003cstrong\u003eA2\u003c/strong\u003e: ESM greenness score; \u003cstrong\u003eB1\u003c/strong\u003e: QAMS blueness applicability score; \u003cstrong\u003eB2\u003c/strong\u003e: ESM blueness applicability score.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7517118/v1/b1b7f9bfe3fc3b98f4dccf76.png"},{"id":91331394,"identity":"fa2b33c3-2e78-4ba3-b19d-6e1f9a9db99e","added_by":"auto","created_at":"2025-09-15 11:04:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":90611,"visible":true,"origin":"","legend":"\u003cp\u003eSingle-factor experiments on processing parameters.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7517118/v1/9d719d606002cb69d03997eb.png"},{"id":91332805,"identity":"e828ff88-fcec-4964-9665-be2ce9d861b4","added_by":"auto","created_at":"2025-09-15 11:20:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":113159,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtraction experiments on steamed \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePanax notoginseng\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7517118/v1/82544131ff0c293aa41fe100.png"},{"id":104250790,"identity":"f2918082-7637-424f-8e23-54356101a85d","added_by":"auto","created_at":"2026-03-09 16:08:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6939766,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7517118/v1/c375eed6-b8ec-4c81-b1f8-fce8cd7337af.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Water-Activation Steaming Coupled with Single-Marker Quantification: A Green Strategy for Industrial Standardization of Bioactive Steamed Panax notoginseng​","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003ePanax notoginseng\u003c/em\u003e (Burk.) F.H.Chen, a \u003cstrong\u003eprized medicinal herb\u003c/strong\u003e in the Araliaceae family, has been extensively applied in traditional Chinese medicine (TCM) with a documented history tracing back to the \u003cem\u003eCompendium of Materia Medica\u003c/em\u003e (\u003cem\u003eBencao Gangmu\u003c/em\u003e) in the Ming Dynasty. Renowned for the principle of \u003cstrong\u003e\u0026quot;raw for dispersion, processed for tonification\u0026quot;\u003c/strong\u003e, its \u003cstrong\u003eraw form\u003c/strong\u003e dissipates stasis and arrests bleeding, while the \u003cstrong\u003esteamed form\u003c/strong\u003e tonifies vitality and strengthens the body\u0026mdash;exemplifying the TCM theory of \u003cstrong\u003edifferential treatment based on processing state\u003c/strong\u003e (\u003cem\u003eSheng Shu Yi Zhi\u003c/em\u003e)[1]. Modern pharmacological studies have confirmed that the primary bioactive constituents of \u003cem\u003eP. notoginseng\u003c/em\u003e are \u003cstrong\u003esaponins\u003c/strong\u003e, with over 150 identified types. These include two main categories: (1) Protopanaxadiol\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003etype (PPD\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003etype) saponins (e.g\u003c/strong\u003e., ginsenosides Rb\u003csub\u003e1\u003c/sub\u003e,\u003csup\u003e\u0026nbsp;\u003c/sup\u003eRd);and (2) Protopanaxatriol\u003cstrong\u003e-type (PPT-type)\u003c/strong\u003e\u003cstrong\u003esaponins\u003c/strong\u003e (e.g., ginsenosides Rg₁, Re)[2-5]. These compounds exert \u003cstrong\u003ehemostatic\u003c/strong\u003e, \u003cstrong\u003eanti-inflammatory\u003c/strong\u003e, and \u003cstrong\u003ecardiovascular protective effects\u003c/strong\u003e by modulating key signaling pathways such as \u003cstrong\u003ePI3K/AKT\u003c/strong\u003e and \u003cstrong\u003eNF-\u0026kappa;B\u003c/strong\u003e[6-8]\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical Transformations During Processing of Raw\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePanax notoginseng\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;and Their Pharmacological Implications\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cstrong\u003ethermal processing\u003c/strong\u003e of raw \u003cem\u003ePanax notoginseng\u003c/em\u003e (Burk.) F.H.Chen triggers a series of \u003cstrong\u003echemical transformations\u003c/strong\u003e that fundamentally alter its \u003cstrong\u003epharmacodynamic basis\u003c/strong\u003e and \u003cstrong\u003epharmacological actions\u003c/strong\u003e, establishing the \u003cstrong\u003ematerial foundation\u003c/strong\u003e for the characteristic \u003cstrong\u003e\u0026quot;differential use of crude and processed forms\u0026quot;\u003c/strong\u003e. High-temperature processing catalyzes \u003cstrong\u003edeglycosylation\u003c/strong\u003e, \u003cstrong\u003edehydration\u003c/strong\u003e, and \u003cstrong\u003eisomerization\u0026nbsp;\u003c/strong\u003eof primary saponins, generating rare ginsenosides with enhanced or unique bioactivities[9-11]. Among these, \u003cstrong\u003eginsenosides Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e, and 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e are established as \u003cstrong\u003ecritical quality markers (CQMs)\u0026nbsp;\u003c/strong\u003efor SPN, with the following scientific rationale:\u003c/p\u003e\n\u003col class=\"decimal_type\" style=\"list-style-type: lower-alpha;\"\u003e\n \u003cli\u003e\u003cstrong\u003eThermal Transformation Specificity: These\u003c/strong\u003e four saponins are the \u003cstrong\u003eprimary degradation products\u003c/strong\u003e formed \u003cem\u003evia\u003c/em\u003e specific pathways under high-temperature/high-humidity steaming (e.g., 105\u0026deg;C, 2~4 h)[12].Their generation follows well-defined routes: Rb\u003cstrong\u003e\u003csub\u003e1\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026rarr;Rd\u0026rarr;Rg\u003csub\u003e3\u003c/sub\u003e\u0026rarr;Rh\u003csub\u003e4\u003c/sub\u003e\u003c/strong\u003e; Rg\u003cstrong\u003e\u003csub\u003e1\u003c/sub\u003e\u0026rarr;Rh\u003csub\u003e1\u003c/sub\u003e\u0026rarr;PPT\u003c/strong\u003e[13]. Their \u003cstrong\u003econcentrations directly correlate\u003c/strong\u003e with processing intensity and efficiency (e.g., Rk\u003csub\u003e3\u003c/sub\u003e content increases 4.7-fold after optimal steaming);\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eDistinct Pharmacological Activities\u003c/strong\u003e\u003cstrong\u003e: Each\u003c/strong\u003e rare ginsenoside contributes uniquely to \u003cstrong\u003etonifying effects\u003c/strong\u003e:\u003cstrong\u003e20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e exhibits \u003cstrong\u003epotent anti-tumor activity\u003c/strong\u003e by inducing apoptosis \u003cem\u003evia\u003c/em\u003e AKT/mTOR inhibition (IC₅₀=12.3\u0026mu;M). \u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e demonstrates \u003cstrong\u003eneuroprotective effects\u003c/strong\u003e, increasing neuron survival by 37.2% in A\u0026beta;₂₅₋₅₅-induced injury models[14]. \u003cstrong\u003eRk₃/Rh₄\u003c/strong\u003esynergistically enhance \u003cstrong\u003eanti-inflammatory\u003c/strong\u003e, \u003cstrong\u003eantioxidant\u003c/strong\u003e, and \u003cstrong\u003ehematopoietic activities\u003c/strong\u003e by activating the PI3K/Akt-Nrf2 axis (\u0026darr;MDA 51%, \u0026uarr;SOD 73%)[9, 12];\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eIndustrial Quality Control (QC) Bottlenecks\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAnalytical reference standards\u003c/strong\u003e for these saponins\u0026mdash;particularly epimers \u003cstrong\u003e20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003eand \u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003eare prohibitively expensive (e.g., \u003cstrong\u003e20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e \u0026asymp; $2,500/mg)[15]. This imposes severe constraints on \u003cstrong\u003eindustrial-scale QC\u003c/strong\u003e when employing \u003cstrong\u003eexternal standard methods (ESM)\u003c/strong\u003e, which require individual reference compounds. The cost significantly limits \u003cstrong\u003ebatch-monitoring frequency\u003c/strong\u003e and \u003cstrong\u003eprocess scalability\u003c/strong\u003e.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eDespite the increasingly clarified material basis for the pharmacological effects of SPN, its industrialization remains constrained by two major technical bottlenecks:\u003c/strong\u003e\u003c/p\u003e\n\u003col style=\"list-style-type: lower-alpha;\"\u003e\n \u003cli\u003e\u003cstrong\u003eProcess\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Variability:\u003c/strong\u003eTraditional processing parameters\u0026mdash;including particle size, water immersion conditions, steaming temperature, and steaming time\u0026mdash;lack systematic optimization based on the kinetics of saponin conversion. Studies indicate that conversion rates are highly sensitive to these parameters[16]. For instance, reducing the particle size from 8 mm to 2 mm increases the yield of Rk3 by 2.2-fold. However, excessive pulverization risks thermal degradation of sensitive constituents. Inadequate water immersion fails to fully activate the conversion pathway [17], while suboptimal temperature-time combinations lead to either incomplete conversion or degradation. These inconsistencies directly result in significant batch-to-batch variations (RSD \u0026gt;15%) in the total content of key rare saponins (e.g., Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e, 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e), critically impacting product efficacy and safety ;\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eLimitations of Current Analytical Methods:\u003c/strong\u003eExisting quality control (QC) primarily relies on the External Standard Method (ESM) for single-component quantification. Although accurate, ESM becomes economically impractical for simultaneously monitoring multiple rare saponins (Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e, 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e, etc.) due to the prohibitively high cost and limited availability of individual reference standards. The Quantitative Analysis of Multi-components by Single Marker (QAMS) approach offers a promising alternative [18, 19]. QAMS quantifies multiple analytes using relative correction factors (RCFs) derived from a single reference standard. However, its application to SPN remains unexplored. A core challenge for QAMS lies in ensuring the robustness and transferability of RCF values. These values are susceptible to significant drift (10%) under variations in chromatographic conditions (e.g., column type). HPLC instrument model, detection wavelength, flow rate), thereby compromising method reliability[20].\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eTo address the aforementioned challenges, this study aims to:\u003c/strong\u003e\u003c/p\u003e\n\u003col style=\"list-style-type: lower-alpha;\"\u003e\n \u003cli\u003e\u003cstrong\u003eOptimize the Steaming Process:\u003c/strong\u003eBy integrating \u003cstrong\u003eorthogonal design\u003c/strong\u003e with \u003cstrong\u003ekinetic modeling\u003c/strong\u003e (based on the identified activation energy for saponin conversion, \u003cem\u003eE\u003c/em\u003ea \u0026asymp; 58.3 kJ/mol )[13], we will develop a stable, efficient, and scalable \u0026quot;\u003cstrong\u003emoisture activation-gradient temperature control\u003c/strong\u003e\u0026quot; production process for SPN. This process targets the \u003cstrong\u003emaximization of key rare saponin yields\u003c/strong\u003e (Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e, 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e) while minimizing thermal degradation risks associated with conventional methods.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eEstablish a Robust QAMS Method:\u003c/strong\u003eWe will develop and validate a novel, cost-effective, and reliable \u003cstrong\u003eHPLC-QAMS method\u003c/strong\u003e using the readily accessible \u003cstrong\u003e20(\u003c/strong\u003e\u003cem\u003eR\u003c/em\u003e\u003cstrong\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e as the internal reference substance\u003c/strong\u003e for simultaneous quantification of four target rare saponins in SPN and its extracts. The study will focus on rigorous evaluation of \u003cstrong\u003erelative correction factor (RCF) stability\u003c/strong\u003e under varied chromatographic conditions (e.g., column type, flow rate, detection wavelength) to ensure method robustness and transferability. Furthermore, the \u003cstrong\u003egreenness and environmental sustainability\u003c/strong\u003e of the proposed method will be comprehensively assessed using \u003cstrong\u003eAnalytical GREEnness (AGREE)\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eBlue Applicability Grade Index (BAGI)\u0026nbsp;\u003c/strong\u003emetrics. These tools will generate quantitative scores based on 12 environmental impact criteria (AGREE) and operational practicality indicators (BAGI), enabling objective comparison with conventional methods.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThis integrated strategy addresses two core challenges-process reproducibility and analytical cost-effectiveness-by establishing a scientifically robust and industrially \u003cem\u003evia\u003c/em\u003eble protocol for the quality standardization of SPN. Consequently, it supports broader clinical and industrial applications while actualizing the value of the \u0026quot;differences in raw and processed products efficacy\u0026quot; theory through modern quality control frameworks.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eReagents, Reference Standards and Sample\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromatography-grade solvents (methanol and acetonitrile, HPLC grade) were purchased\u003c/strong\u003e from Fisher Scientific (Fair Lawn, NJ, USA). \u003cstrong\u003eUltrapure water\u003c/strong\u003e was prepared using a Milli-Q system (Millipore, Bedford, MA, USA) from purified water sourced from Wahaha Group (Hangzhou, China). Ginsenoside\u003cstrong\u003e\u0026nbsp;reference standards\u003c/strong\u003e\u003cstrong\u003e: Rk\u003c/strong\u003e\u003csub\u003e3\u003c/sub\u003e and Rh\u003csub\u003e4\u003c/sub\u003e were obtained from Stanford Analytical Chemicals Inc. (Stanford, CA, USA); 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e was supplied by Chengdu GLP Biotechnology Co., Ltd. (Chengdu, China);20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e was procured from the National Institutes for Food and Drug Control (NIFDC, Beijing, China). \u003cem\u003ePurity\u003c/em\u003e\u003cem\u003e\u0026nbsp;validation\u003c/em\u003e: All standards were verified by \u003cstrong\u003eHPLC peak area normalization\u003c/strong\u003e, with purities exceeding 98% (Table\u0026nbsp;1). General\u003cstrong\u003e\u0026nbsp;chemicals\u003c/strong\u003e: Analytical-grade reagents were purchased from Beijing Chemical Works (Beijing, China). A total of \u003cstrong\u003e15\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ebatches of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSPN\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;powder\u003c/strong\u003e were collected from five manufacturers in China. All samples were produced by \u003cstrong\u003eslicing and steaming raw roots\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003esourced from Yunnan Province (Table 2). Sample authentication was performed by macroscopic and microscopic examination according to the Chinese Pharmacopoeia (2025 Edition).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Reference material information\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eName\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eProducer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eModel No.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBatch No.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eConcentration\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCAS NO.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRk\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eStanford Analytical Chemicals Inc.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eLDHT-1020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePT231207-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.76%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e174721-08-5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRh\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eStanford Analytical Chemicals Inc.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eEAAF-2029\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBE231103-21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.97%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e364779-15-7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e20（\u003cem\u003eS\u003c/em\u003e）-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eChengdu Glip Biotechnology Co.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eJOT-10044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e24021802\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.39%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14197-60-5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e20（\u003cem\u003eR\u003c/em\u003e）-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eChina Academy of Food and Drug Control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e110804\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e201504\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99.50%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e38243-03-7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e SPN powder herbal pieces sample information\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBatch No.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSource/Manufacturer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eProcessing Method\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSY-WS20230201\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eYunnan Baiyao Group Sanqi Industrial Co., Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSliced notoginseng steaming\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSY-WS20230202\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eYunnan Baiyao Group Sanqi Industrial Co., Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSliced notoginseng steaming\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSY-WS20230203\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eYunnan Baiyao Group Sanqi Industrial Co., Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSliced notoginseng steaming\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSY-WS20230204\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eYunnan Baiyao Group Sanqi Industrial Co., Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSliced notoginseng steaming\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSY-WS20230205\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eYunnan Baiyao Group Sanqi Industrial Co., Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSliced notoginseng steaming\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSY-WS20230206\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eYunnan Baiyao Group Sanqi Industrial Co., Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSliced notoginseng steaming\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eY202302001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eYunnan Qidan Pharmaceutical Co., Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSliced notoginseng steaming\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eY202302002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eYunnan Qidan Pharmaceutical Co., Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSliced notoginseng steaming\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eY202302003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eYunnan Qidan Pharmaceutical Co., Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSliced notoginseng steaming\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eY202302004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eYunnan Qidan Pharmaceutical Co., Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSliced notoginseng steaming\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eY202302005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eYunnan Qidan Pharmaceutical Co., Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSliced notoginseng steaming\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eY202302006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eYunnan Qidan Pharmaceutical Co., Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSliced notoginseng steaming\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eH20230404\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eYunnan Baiyao Group TCM Resources Co., Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSliced notoginseng steaming\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eH20230405\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eYunnan Baiyao Group TCM Resources Co., Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSliced notoginseng steaming\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eH20230406\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eYunnan Baiyao Group TCM Resources Co., Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSliced notoginseng steaming\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eStandard solutions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA mixed standard stock solution containing ginsenosides\u0026nbsp;Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e, 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e was prepared in methanol. Serial dilutions of this stock solution were performed using 60% (v/v) ethanol to obtain working solutions, with concentrations of 0.1557, 0.1561, 0.1472, and 0.0856 mg/mL for Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, 20(S)-Rg\u003csub\u003e3\u003c/sub\u003e, and 20(R)-Rg\u003csub\u003e3\u003c/sub\u003e, respectively. Prior to HPLC analysis, all diluted solutions were filtered through 0.22 \u0026mu;m microporous membranes (Millipore, Billerica, MA, USA). The filtrates were injected into the HPLC system, and calibration curves were constructed by plotting peak areas against respective concentrations (Table 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Preparation concentrations of reference standard solutions\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eName\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eConcentration(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003eWeighed Amount (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eStock Solution Conc.（mg/mL）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003eWorking Solution Conc. (mg/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eRk\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e98.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e39.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e1.5570\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e0.1557\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eRh\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e98.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e39.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e1.5605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e0.1561\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e98.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e37.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e1.4719\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e0.1472\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e99.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e17.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e0.17114\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e0.0856\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eSample Preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSieving \u0026amp; Moistening\u003c/strong\u003e: SPN powder was sieved through a \u003cstrong\u003eNo. 4 sieve\u003c/strong\u003e (nominal aperture: 4.0 mm; dominant particle size: 2~4 mm). The sieved powder was moistened with \u003cstrong\u003epurified water\u003c/strong\u003e (equivalent to its own weight) and allowed to equilibrate for \u003cstrong\u003e2 hours. Steaming \u0026amp; Drying\u003c/strong\u003e: The moistened sample was steamed at \u003cstrong\u003e120\u0026deg;C for 5 hours\u003c/strong\u003e in a saturated steam environment, followed by natural cooling and air-drying to ambient temperature. \u003cstrong\u003eGrinding \u0026amp; Weighing\u003c/strong\u003e: The dried material was re-ground through the No. 4 sieve. Approximately \u003cstrong\u003e0.6 g\u003c/strong\u003e of the powder was accurately weighed (precision: \u0026plusmn;0.1 mg) into a round-bottom flask. \u003cstrong\u003eReflux Extraction\u003c/strong\u003e:\u003cstrong\u003e50 mL of 60% (v/v) ethanol\u003c/strong\u003e was added, and the total weight was recorded. The mixture underwent \u003cstrong\u003ereflux extraction\u003c/strong\u003e at \u003cstrong\u003e80\u0026deg;C for 1 hour\u003c/strong\u003e using a water bath. \u003cstrong\u003eCooling \u0026amp; Replenishment: After\u003c/strong\u003e cooling to room temperature, the weight loss due to solvent evaporation was compensated by adding fresh 60% ethanol. The mixture was vortex-mixed for homogeneity. \u003cstrong\u003eFiltration: The\u003c/strong\u003e extract was filtered through a \u003cstrong\u003e0.22 \u0026mu;m nylon membrane filter\u003c/strong\u003e (Millipore, Billerica, MA, USA) to remove particulate matter prior to HPLC injection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHPLC analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromatographic system\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInstrumentation: Analysis was performed on an Agilent 1260 Infinity II LC system equipped with: Quaternary Pump (G7131C, pressure range up to 600 bar), Autosampler with thermostat-controlled sample tray, Column Compartment (temperature accuracy: \u0026plusmn;0.5\u0026deg;C), Diode Array Detector (DAD) with high-sensitivity flow cell (G7117B). Chromatographic Conditions: Column Agilent ZORBAX SB-C18 (250 mm \u0026times; 4.6 mm, 5 \u0026mu;m particle size); Mobile phase Acetonitrile-water (40 : 60, v/v), isocratic elution; Flow rate 1.0 mL/min; Column temperature 25\u0026deg;C; Detection wavelength 203 nm; Injection volume 10 \u0026mu;L. System suitability: Theoretical plates for 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u0026ge; 6000, Sample Analysis: Chromatograms of reference standards (Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e, 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e) and SPN\u003cem\u003e\u0026nbsp;\u003c/em\u003eextracts are provided in Figure 1 (\u003cstrong\u003eHPLC chromatograms of the mixed reference solution (A) and steamed\u0026nbsp;Panax notoginseng\u0026nbsp;extract (B)\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ePeaks1: Rk\u003csub\u003e3\u003c/sub\u003e; 2: Rh\u003csub\u003e4\u003c/sub\u003e; 3: 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e; 4: 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e. Chromatographic conditions: Agilent ZORBAX-SB-C18 column (250 mm \u0026times; 4.6 mm, 5 \u0026mu;m); isocratic elution with acetonitrile-water (40:60, v/v); flow rate 1.0 mL/min; column temperature 25\u0026deg;C; detection wavelength 203 nm; injection volume 10 \u0026mu;L).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation of the HPLC Analytical Method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rationality of the HPLC analytical method was verified through assessments of \u003cstrong\u003especificity, linearity and range, precision, stability, repeatability, accuracy, and robustness\u003c/strong\u003e. The validation procedures were designed as follows: Precision\u003cstrong\u003e\u0026nbsp;(Intra-day)\u003c/strong\u003e: The same test solution was analyzed \u003cstrong\u003esix times consecutively within one day\u003c/strong\u003e to evaluate intra-day precision; \u003cstrong\u003eRepeatability (Intermediate Precision)\u003c/strong\u003e: Six independent sample preparations from the same batch were analyzed in parallel to assess repeatability (intermediate precision); \u003cstrong\u003eAccuracy\u003c/strong\u003e: Accuracy was determined by \u003cstrong\u003estandard\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eaddition recovery experiments\u003c/strong\u003e. A known amount of reference standard (1:1 ratio to the target analyte) was spiked into the test solution, and the \u003cstrong\u003erecovery rate (%)\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewas calculated; \u003cstrong\u003eSolution Stability\u003c/strong\u003e: The stability of the sample solution was evaluated at \u003cstrong\u003e0, 2, 4, 8, 12, 16, 20, 24, and 36 hours\u003c/strong\u003e post-preparation under controlled storage conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod validation for QAMS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRelative correction factor\u003c/strong\u003e\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eStandard Solution Preparation:\u0026nbsp;\u003c/strong\u003eAliquots (2, 4, 6, 8, 10, and 12 \u0026mu;L) of a mixed reference standard solution containing \u003cstrong\u003eginsenosides Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e, and 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e were precisely withdrawn and injected into the HPLC system under the chromatographic conditions specified in \u003cstrong\u003eSection\u0026nbsp;\u003c/strong\u003eChromatographic system.\u003c/h4\u003e\n\u003cp\u003e\u003cstrong\u003eCalculation of Relative Correction Factors (RCFs):\u0026nbsp;\u003c/strong\u003eUsing \u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e as the internal reference substance, the RCF for each target analyte (mi= Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, or 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e) was calculated according to \u003cstrong\u003eEquation (1)\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"396\" height=\"79\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere: \u003cem\u003eF\u003c/em\u003e\u003csub\u003e20(\u003cem\u003eR\u003c/em\u003e)Rg3/mi:\u003c/sub\u003e Relative correction factor of the target analyte (mi) to \u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e; \u003cem\u003eA\u003c/em\u003e\u003csub\u003e20(\u003cem\u003eR) Rg\u003c/em\u003e3\u003c/sub\u003e and Ami: Peak areas of \u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e and the target analyte (mi), respectively; \u003cem\u003eC\u003c/em\u003e\u003csub\u003e20(\u003cem\u003eR) Rg\u003c/em\u003e3\u003c/sub\u003e and \u003cem\u003eC\u003csub\u003emi:\u0026nbsp;\u003c/sub\u003e\u003c/em\u003eConcentrations (\u0026mu;g/mL) of \u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e and the target analyte (mi) in the mixed standard solution, respectively. \u003cem\u003eRCF validation\u003c/em\u003e: Each RCF value was derived from \u003cstrong\u003esix-point calibration curves\u003c/strong\u003e and validated through triplicate injections.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQAMS for Saponin Content Determination in SPN\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Content by External Standard Method (ESM): The\u003c/strong\u003e content of\u0026nbsp;\u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e in 15 batches of SPN was determined using a \u003cstrong\u003ecalibration curve\u003c/strong\u003e derived from the reference standard.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQAMS-Based Calculation of Target Saponin Concentrations:\u0026nbsp;\u003c/strong\u003eThe concentrations of \u003cstrong\u003eginsenosides Rk3, Rh4, and 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e were calculated according to \u003cstrong\u003eEquation (2)\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"320\" height=\"60\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere:\u003c/p\u003e\n\u003cp\u003eC\u003csub\u003ei\u003c/sub\u003e and A\u003csub\u003ei\u003c/sub\u003e = \u003cstrong\u003eConcentration (mg/mL)\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003epeak area\u003c/strong\u003e of the target analyte (Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, or 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e);\u003c/p\u003e\n\u003cp\u003eC\u003csub\u003es\u003c/sub\u003e and A\u003csub\u003es\u0026nbsp;\u003c/sub\u003e= \u003cstrong\u003eConcentration (mg/mL)\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003epeak area\u003c/strong\u003e of the internal reference substance\u0026nbsp;\u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e;\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"65\" height=\"21\" src=\"data:image/png;base64,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\" alt=\"image\"\u003e = \u003cstrong\u003eRelative correction factor (RCF)\u0026nbsp;\u003c/strong\u003eof the target analyte to \u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e (validated in Section 3.2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContent Calculation of Target Saponins:\u0026nbsp;\u003c/strong\u003eThe content of each target saponin in steamed \u003cem\u003eP. notoginseng\u003c/em\u003e was calculated using \u003cstrong\u003eEquation (3)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"232\" height=\"70\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere:\u003c/p\u003e\n\u003cp\u003eR\u003csub\u003ei\u003c/sub\u003e = \u003cstrong\u003eContent (mg/g) \u003c/strong\u003eof the target analyte (Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, or 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e);\u003c/p\u003e\n\u003cp\u003eC\u003csub\u003ei\u003c/sub\u003e = \u003cstrong\u003eConcentration (mg/mL) \u003c/strong\u003ecalculated from Equation (2);\u003c/p\u003e\n\u003cp\u003eV = \u003cstrong\u003eVolume of sample solution (50 mL)\u003c/strong\u003e;\u003c/p\u003e\n\u003cp\u003eM = \u003cstrong\u003eSample mass (0.6000 g) \u003c/strong\u003eaccurately weighed during preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of the proposed method\u0026apos;s greenness\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe greenness assessment was performed using the Analytical GREEnness (AGREE) metric[21]. This method establishes an evaluation system based on the twelve core principles of Green Analytical Chemistry (GAC). It employs a weighted calculation to derive a composite score ranging from 0 to 1 (in standardized units), which is visualized as a circular pictogram with the integrated score displayed at its center. The AGREE assessment tool utilized in this study is accessible \u003cem\u003evia\u003c/em\u003e reference 23. Additionally, the Blue Applicability Grade Index (BAGI) method [22-24] was introduced to evaluate the functionality and practical utility of the analytical approach[25].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDevelopment of Extraction Methods for SPN Saponins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOrthogonal Experimental Design for Extraction Processes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe extraction parameters (ethanol concentration, solid-to-liquid ratio, extraction time, and extraction frequency) were optimized using an L9(3⁴) orthogonal array design, with the transfer rate of 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e and total extract yield as evaluation metrics[26, 27]. SPN granules (30g per batch, 9 batches total) underwent reflux extraction under conditions specified in Table 4. Each extract was diluted to 300 mL, and a 300 mL aliquot was dried to calculate the yield; combined extracts were concentrated, dried, and analyzed for total saponin content. Key influencing factors were identified \u003cem\u003evia\u003c/em\u003e range analysis and analysis of variance (ANOVA).\u003c/p\u003e\n\u003ch3\u003eTable 4. \u003cem\u003eL\u003c/em\u003e\u003cstrong\u003e9(3\u003c/strong\u003e\u003cstrong\u003e^\u003c/strong\u003e\u003cstrong\u003e4) Orthogonal \u003c/strong\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003cstrong\u003experimental \u003c/strong\u003e\u003cstrong\u003el\u003c/strong\u003e\u003cstrong\u003eayout for \u003c/strong\u003e\u003cstrong\u003et\u003c/strong\u003e\u003cstrong\u003eotal Saponins \u003c/strong\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003cstrong\u003extraction from \u003c/strong\u003e\u003cstrong\u003eSPN\u003c/strong\u003e\u003c/h3\u003e\n\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTrial\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eEthanol Concentration (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSolid/Liquid Ratio (-fold)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eReflux Time (h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eExtraction Cycles\u003c/p\u003e\n \u003cp\u003e(n)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e50%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5(1:5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e50%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.5(1:7.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e50%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10(1:10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e60%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5(1:5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e60%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.5(1:7.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e60%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10(1:10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e70%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5(1:5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e70%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.5(1:7.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e70%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10(1:10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eOptimization of Key Parameters in Extraction Processes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the orthogonal experimental results, a single-factor experiment was conducted to optimize extraction frequency. Three batches of SPN granules (30 g each) were reflux-extracted with 7.5 volumes of 60% ethanol for 1.5 hours per cycle, with frequencies of 4, 5, and 6 times (each condition in duplicate). The combined extracts were filtered, concentrated under reduced pressure to recover ethanol, and lyophilized to powder. The \u003cstrong\u003etotal extract yield\u003c/strong\u003e was calculated as the ratio of dried extract mass to initial sample mass \u0026times; 100%. The \u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e content\u003c/strong\u003e was quantified \u003cem\u003evia\u003c/em\u003e HPLC-DAD, and its \u003cstrong\u003etransfer rate\u003c/strong\u003e was determined by the ratio of \u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e mass in the extract to that in the raw material \u0026times; 100%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and Purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo obtain a saponin extract with higher purity and enriched rare ginsenosides, the mixed extract from Section Optimization of Key Parameters in Extraction Processes underwent further purification. Based on preliminary studies, the optimized protocol comprised: combining extracts from five cycles, concentrating under reduced pressure to a density of 1.1 g/mL, and purifying \u003cem\u003evia\u003c/em\u003e D101 macroporous resin (loading concentration: 0.2 g/mL; flow rate: 0.5 BV/h)[28-30]. Sequential elution with 4BV deionized water and 4BV 100% ethanol was followed by decolorization using D941 anion-exchange resin (column height-to-diameter ratio: 10:1; flow rate: 1 BV/h)[31]. The decolorized solution was rinsed with 1.5 BV of 68% ethanol, and the combined eluates were spray-dried (inlet temperature: 265 \u0026plusmn; 10\u0026deg;C; outlet: 80 \u0026plusmn; 5\u0026deg;C; pressure \u0026ge;0.4 MPa)[32]. The resulting powder was sieved through 120-mesh, homogenized for 30 min, and stored in light-proof containers under \u0026lt;30% humidity[33].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of Extracts from SPN\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder the guidance of the optimized extraction process, \u003cstrong\u003elarge-scale production of SPN extracts\u003c/strong\u003e was conducted in collaboration with \u003cstrong\u003eYunnan Baiyao Group Wenshan QiHua Co., Ltd. (Wenshan, China)\u003c/strong\u003e. Three batches of extracts (Batch Nos.: S20230401, S20231113, S20240201) were prepared. The \u003cstrong\u003emoisture content\u003c/strong\u003e of the extracts was determined according to the \u003cstrong\u003edrying loss method\u003c/strong\u003e specified in \u003cstrong\u003ePart 0832 of the Chinese Pharmacopoeia (2020 Edition)\u003c/strong\u003e. Quantification of four target ginsenosides Rk\u003csub\u003e3, \u003c/sub\u003eRh\u003csub\u003e4\u003c/sub\u003e,20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e, and 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3 \u003c/sub\u003ewas performed using a \u003cstrong\u003evalidated Quantitative Analysis of Multi-Components by Single Marker (QAMS).\u003c/strong\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMethod Validation for HPLC Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLinearity and Range\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe reference standards of\u003cstrong\u003e\u0026nbsp;Rk\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Rh₄, and 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e (40 mg each) were accurately weighed and separately dissolved in methanol in 25 mL volumetric flasks to prepare stock solutions with concentrations of \u003cstrong\u003e1.5570 mg/mL (Rk\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e, \u003cstrong\u003e1.5605\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emg/mL (Rh\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e4\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e, and \u003cstrong\u003e1.4719\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emg/mL (20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e. Separately, \u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e (20 mg) was dissolved in methanol in a 100 mL volumetric flask, yielding a stock solution at \u003cstrong\u003e0.17114 mg/mL\u003c/strong\u003e. Aliquots (1mL each) of the \u003cstrong\u003eRk\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e, \u003cstrong\u003eRh\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e4\u003c/sub\u003e\u003c/strong\u003e, and \u003cstrong\u003e20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e stock solutions were transferred to a 10 mL volumetric flask, mixed with \u003cstrong\u003e5mL of 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;stock solution\u003c/strong\u003e, and diluted to volume with \u003cstrong\u003e60% ethanol\u003c/strong\u003e, resulting in a mixed working solution with final concentrations of \u003cstrong\u003e0.1557\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emg/mL(Rk\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e, \u003cstrong\u003e0.1561\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emg/mL\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(Rh\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e4\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e, \u003cstrong\u003e0.1472\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emg/mL\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e, and \u003cstrong\u003e0.0856\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emg/mL\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e.\u0026nbsp;Six injections (2, 4, 6, 8, 10, and 12\u0026nbsp;\u0026mu;L) of the mixed solution were analyzed by HPLC, establishing linear relationships between injection volume (\u003cem\u003eX\u003c/em\u003e, \u0026mu;L) and peak area (\u003cem\u003eY\u003c/em\u003e) for all four ginsenosides. Regression analysis confirmed excellent linearity with \u003cstrong\u003edetermination coefficients (R\u0026sup2;) \u0026gt; 0.9997\u003c/strong\u003e; detailed regression data are summarized in \u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e5\u003c/strong\u003e, and standard curves are illustrated in \u003cstrong\u003eFigure\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2 (\u003c/strong\u003e\u003cstrong\u003eStandard curves of four ginsenosides: Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e, and 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 5. \u003cstrong\u003eCalibration\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003cstrong\u003eurves for\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003cstrong\u003eour\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003cstrong\u003eharacteristic Saponins in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSPN\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003eName\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 176px;\"\u003eRegression Equation\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003eLinear Rang（\u0026mu;g）\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003eR\u003csup\u003e2\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003eRk\u003csub\u003e3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 176px;\"\u003ey = 653.2x - 4.813\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e0.3114～1.8684\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e1.0000\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003eRh\u003csub\u003e4\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 176px;\"\u003ey = 803.6x - 6.187\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e0.3121～1.8726\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e1.0000\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 176px;\"\u003ey = 334.5x - 1.818\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e0.2944～1.7663\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e0.9999\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 176px;\"\u003ey = 376.2x - 1.391\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e0.1711～1.7114\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e0.9997\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003ePrecision (Repeatability), Stability, and Accuracy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe method validation results, expressed as \u003cstrong\u003erelative standard de\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003evia\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003etions (RSD) of chromatographic peak areas\u003c/strong\u003e, demonstrate high reliability, with lower RSD values indicating superior precision. For the \u003cstrong\u003erepeatability assessment\u003c/strong\u003e, six replicate analyses of the same sample yielded RSD values below \u003cstrong\u003e0.38%\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efor all target peaks. \u003cstrong\u003eSample stability\u003c/strong\u003e was confirmed by RSD values \u0026le; \u003cstrong\u003e1.2%\u003c/strong\u003e across peak areas measured at multiple time points over \u003cstrong\u003e36 h at room temperature\u003c/strong\u003e. The \u003cstrong\u003emethod reproducibility\u003c/strong\u003e was verified using six independently prepared samples from the same batch, showing RSD values \u0026lt; \u003cstrong\u003e1.0%\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efor all peaks. \u003cstrong\u003eAccuracy\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewas evaluated \u003cem\u003evia\u003c/em\u003e \u003cstrong\u003espike recovery tests\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewith known concentrations of reference standards, achieving recoveries of \u003cstrong\u003e91.95\u0026ndash;101.34%\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efor four major saponins in SPN, with associated RSDs ranging from \u003cstrong\u003e0.93% to 1.8%\u003c/strong\u003e. Collectively, these data validate the method\u0026apos;s robustness and reliability; detailed results are provided in \u003cstrong\u003eTable\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;6\u003c/strong\u003e\u003cimg width=\"4\" height=\"19\" src=\"data:image/png;base64,R0lGODdhBAATAHcAACH+GlNvZnR3YXJlOiBNaWNyb3NvZnQgT2ZmaWNlACwAAAAABAATAIIAOY6OOQDY/Pz8/Nj8/PwBAgMBAgMBAgMDC0i63P4wSjICELMlADs=\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003eTable 6. \u003cstrong\u003eMethod\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ev\u003c/strong\u003e\u003cstrong\u003ealidation for HPLC\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003enalysis of Saponins in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSPN\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003eComposition\u003cbr\u003e\u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003ePrecision RSD (%)\u003cbr\u003e\u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003eStability RSD (%)\u003cbr\u003e\u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003eRepeatability RSD (%)\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\"\u003eAccuracy\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003eRecovery Rate (%)\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003eRSD (%)\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003eRk\u003csub\u003e3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.10\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.34\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.46\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e93.98\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.40\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003eRh\u003csub\u003e4\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.07\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.18\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.37\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e94.26\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.80\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.27\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.47\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.75\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e91.95\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.89\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.38\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.20\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.90\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e101.34\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.93\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eValidation and Application of a QAMS-Based Chemometric Model for Multi-Component Quantitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn this study, ginsenoside 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;was identified as the most stable and readily accessible saponin in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSPN\u003c/strong\u003e\u003cstrong\u003e. It exhibits excellent separation under conventional chromatographic conditions with mature isolation techniques. Additionally, its relatively low acquisition cost and significant bioactivities\u0026mdash;including anticancer, immunomodulatory, antioxidant, anti-inflammatory, and cardiovascular protective effects\u0026mdash;collectively establish 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;as the optimal internal standard for content determination in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSPN\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRelative Correction Factor (RCF)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWithin the validated linearity range, the content of saponins exhibited a proportional relationship with chromatographic peak areas. Variations in injection volumes simultaneously altered both saponin content and detector responses; however, the relative correction factors (RCFs) theoretically remained constant. The mean RCF values calculated across different injection volumes were adopted as final results, with their relative standard de\u003cem\u003evia\u003c/em\u003etions (RSDs) reflecting computational stability. To ensure precision, all RCFs were uniformly retained to four decimal places. The averaged RCFs for \u003cstrong\u003eginsenosides Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, and 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u0026nbsp;\u003c/strong\u003ewere determined as \u003cstrong\u003e0.5768, 0.4690, and 1.0924\u003c/strong\u003e, respectively, with a maximum RSD of \u003cstrong\u003e1.1%\u0026nbsp;\u003c/strong\u003e\u0026mdash;significantly below the 2.0% threshold\u0026mdash;confirming robust stability of the calculated RCFs. Comprehensive results are detailed in \u003cstrong\u003eTable 7\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTable 7. \u003cstrong\u003eRelative Correction Factors (\u003c/strong\u003e\u003cem\u003eF\u003c/em\u003e\u003cstrong\u003e) of Saponins in\u0026nbsp;\u003c/strong\u003e\u003cem\u003ePanax notoginseng\u003c/em\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003eInjection Volume (\u0026mu;L)\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003eF\u003csub\u003eRk3/20\u003c/sub\u003e\u003csub\u003e（\u003c/sub\u003e\u003cem\u003e\u003csub\u003eR\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e）\u003c/sub\u003e\u003csub\u003e-Rg3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003eF\u003csub\u003eRh4\u003c/sub\u003e\u003csub\u003e/20\u003c/sub\u003e\u003csub\u003e（\u003c/sub\u003e\u003cem\u003e\u003csub\u003eR\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e）\u003c/sub\u003e\u003csub\u003e-Rg3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003eF\u003csub\u003e20\u003c/sub\u003e\u003csub\u003e（\u003c/sub\u003e\u003cem\u003e\u003csub\u003eS\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e）\u003c/sub\u003e\u003csub\u003e-Rg3/20\u003c/sub\u003e\u003csub\u003e（\u003c/sub\u003e\u003cem\u003e\u003csub\u003eR\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e）\u003c/sub\u003e\u003csub\u003e-Rg3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e12\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.5740\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.4665\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.0896\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e10\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.5818\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.4731\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.1003\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e8\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.5739\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.4670\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.0860\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e6\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.5746\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.4666\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.0795\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e4\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.5751\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.4685\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.0877\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e2\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.5815\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.4721\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.1113\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003eMean\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.5768\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.4690\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.0924\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 122px;\"\u003eRSD(%)\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e0.65\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e0.62\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e1.10\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eRobustness Testing and Evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe influence of \u003cstrong\u003eflow rate variations (0.9, 1.0, and 1.1 mL/min)\u003c/strong\u003e, \u003cstrong\u003edetection wavelengths (202, 203, and 204\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003enm)\u003c/strong\u003e, \u003cstrong\u003eHPLC systems (Thermo Fisher Ultimate 3000, Shimadzu LC-20AD, Agilent 1260 Infinity)\u003c/strong\u003e, and \u003cstrong\u003echromatographic columns [Agilent ZORBAX-C18 (4.6\u0026times;250 mm, 5\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026mu;m), Shimadzu VP-ODS (4.6\u0026times;250\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emm, 5\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026mu;m), Inertsil ODS-3 C18 (4.6\u0026times;250\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emm, 5\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026mu;m)]\u003c/strong\u003e on the relative correction factors (RCFs) of ginsenosides \u003cstrong\u003eRk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, and 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e in the QAMS method was systematically evaluated. Results demonstrated excellent stability of RCFs across all conditions: \u003cstrong\u003eflow rate variations\u003c/strong\u003e yielded RSD \u0026lt; 1.0%, \u003cstrong\u003ewavelength adjustments\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eshowed RSD \u0026lt; 1.3%, and \u003cstrong\u003einter-system/column variations\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eexhibited RSD \u0026lt; 2.0% (Table\u0026nbsp;8). All de\u003cem\u003evia\u003c/em\u003etions were within acceptable limits\u0026nbsp;(5%), confirming the method\u0026apos;s robustness for routine application in diverse laboratory environments.\u003c/p\u003e\n\u003cp\u003eTable 8. \u003cstrong\u003eRobustness\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003cstrong\u003evaluation of Relative Correction Factors (\u003c/strong\u003e\u003cem\u003eF\u003c/em\u003e\u003cstrong\u003e) for Saponins\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eu\u003c/strong\u003e\u003cstrong\u003ender\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ev\u003c/strong\u003e\u003cstrong\u003earied HPLC\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003cstrong\u003eonditions\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"626\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003eInstrument\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003eFlow Rate (mL/min)\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003eF\u003csub\u003eRk3/20\u003c/sub\u003e\u003csub\u003e（\u003c/sub\u003e\u003cem\u003e\u003csub\u003eR\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e）\u003c/sub\u003e\u003csub\u003e-Rg3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003eF\u003csub\u003eRh4\u003c/sub\u003e\u003csub\u003e/20\u003c/sub\u003e\u003csub\u003e（\u003c/sub\u003e\u003cem\u003e\u003csub\u003eR\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e）\u003c/sub\u003e\u003csub\u003e-Rg3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003eF\u003csub\u003e20\u003c/sub\u003e\u003csub\u003e（\u003c/sub\u003e\u003cem\u003e\u003csub\u003eS\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e）\u003c/sub\u003e\u003csub\u003e-Rg3/20\u003c/sub\u003e\u003csub\u003e（\u003c/sub\u003e\u003cem\u003e\u003csub\u003eR\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e）\u003c/sub\u003e\u003csub\u003e-Rg3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 175px;\"\u003eShimadzu LC-20AD\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e0.9\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e0.5715\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e0.4718\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e1.0727\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e1.0\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e0.5799\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e0.4722\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e1.0751\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e1.1\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e0.5713\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e0.4651\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e1.0926\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 321px;\"\u003eMean\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e0.5742\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e0.4697\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e1.0801\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 321px;\"\u003eRSD%\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e0.86\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e0.85\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e1.10\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003eInstrument\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003eWavelength (nm)\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003eF\u003csub\u003eRk3/20\u003c/sub\u003e\u003csub\u003e（\u003c/sub\u003e\u003cem\u003e\u003csub\u003eR\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e）\u003c/sub\u003e\u003csub\u003e-Rg3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003eF\u003csub\u003eRh4\u003c/sub\u003e\u003csub\u003e/20\u003c/sub\u003e\u003csub\u003e（\u003c/sub\u003e\u003cem\u003e\u003csub\u003eR\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e）\u003c/sub\u003e\u003csub\u003e-Rg3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003eF\u003csub\u003e20\u003c/sub\u003e\u003csub\u003e（\u003c/sub\u003e\u003cem\u003e\u003csub\u003eS\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e）\u003c/sub\u003e\u003csub\u003e-Rg3/20\u003c/sub\u003e\u003csub\u003e（\u003c/sub\u003e\u003cem\u003e\u003csub\u003eR\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e）\u003c/sub\u003e\u003csub\u003e-Rg3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 175px;\"\u003eShimadzu LC-20AD\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e202\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e0.5753\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e0.4732\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e1.0672\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e203\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e0.5799\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e0.4769\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e1.0751\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e204\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e0.5809\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e0.4652\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e1.0820\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 321px;\"\u003eMean\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e0.5787\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e0.4718\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e1.0748\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 321px;\"\u003eRSD%\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e0.52\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e1.30\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e0.69\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003eHPLC\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003eColumn Type\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003eF\u003csub\u003eRk3/20\u003c/sub\u003e\u003csub\u003e（\u003c/sub\u003e\u003cem\u003e\u003csub\u003eR\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e）\u003c/sub\u003e\u003csub\u003e-Rg3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003eF\u003csub\u003eRh4\u003c/sub\u003e\u003csub\u003e/20\u003c/sub\u003e\u003csub\u003e（\u003c/sub\u003e\u003cem\u003e\u003csub\u003eR\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e）\u003c/sub\u003e\u003csub\u003e-Rg3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003eF\u003csub\u003e20\u003c/sub\u003e\u003csub\u003e（\u003c/sub\u003e\u003cem\u003e\u003csub\u003eS\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e）\u003c/sub\u003e\u003csub\u003e-Rg3/20\u003c/sub\u003e\u003csub\u003e（\u003c/sub\u003e\u003cem\u003e\u003csub\u003eR\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e）\u003c/sub\u003e\u003csub\u003e-Rg3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003eThermofisher Ultimate 3000\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003eAgilent ZORBAX-C18\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e0.5733\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e0.4662\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e1.0856\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003eShimadzu LC-20AD\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003eShim-pack VP-ODS\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e0.5712\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e0.4757\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e1.0450\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003eAngilent 1260 Infinity\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003eInertsil ODS-3 C18\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e0.5848\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e0.4744\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e1.0631\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 321px;\"\u003eMean\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e0.5764\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e0.4721\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e1.0646\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 321px;\"\u003eRSD%\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e1.30\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e1.10\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e2.00\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;Agreement Assessment between QAMS and External Standard Method\u003c/p\u003e\n\u003cp\u003eTo validate the reliability of the QAMS method, this study conducted parallel quantification of four saponins in 15 batches of \u003cem\u003eSPN\u003c/em\u003e using both QAMS and the external standard method (ESM), in accordance with \u003cem\u003eChinese Pharmacopoeia\u003c/em\u003e General Rule 9101. The consistency between methods was assessed \u003cem\u003evia\u003c/em\u003e \u003cstrong\u003eRelative Error (RE%)\u003c/strong\u003e, calculated as: RE% = [(QAMS \u0026minus; ESM)/EMS] \u0026times; 100%. As shown in Table 9, the mean RE% values for \u003cstrong\u003eginsenosides Rk₃, Rh₄, and 20(S)-Rg₃\u003c/strong\u003e were \u003cstrong\u003e-0.18%\u003c/strong\u003e, \u003cstrong\u003e0.83%\u003c/strong\u003e, and\u003cstrong\u003e0.87%\u003c/strong\u003e, respectively, with all absolute RE% values below \u003cstrong\u003e5%\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u0026mdash; meeting the acceptance criteria for method comparability per ICH Q2(R1) guidelines. These results align with the validation conclusions of Chen et al. in red ginseng multi-component analysis (RE% \u0026lt; 4.8%) , confirming the accuracy and stability of the established QAMS method. Crucially, QAMS \u003cstrong\u003ereduced consumption of expensive reference standards (notably\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e20(S)-Rg₃) by 75%\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewhile demonstrating exceptional robustness (RSD \u0026lt; 3.5%) across three HPLC systems (ThermoFisher Ultimate 3000, Shimadzu LC-20AD, Agilent 1260 Infinity) and multiple C18 columns. This robustness ensures method transferability between laboratories and instrument platforms, generating highly comparable and reliable data. By significantly lowering barriers to industrial-scale multi-component monitoring (due to scarce/expensive standards) and reducing analytical costs, QAMS provides a robust technical foundation for establishing a unified, standardized quality evaluation system for\u0026nbsp;SPN\u0026nbsp;and its preparations \u0026mdash; a critical step toward intelligent manufacturing and end-to-end quality control in traditional Chinese medicine.\u003c/p\u003e\n\u003cp\u003eTable 9. \u003cstrong\u003eQuantification of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003cstrong\u003eour Saponins in 15\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003eatches of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSPN\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;by ESM and QAMS\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003em\u003c/strong\u003e\u003cstrong\u003eethods\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"104%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 10px;\"\u003eSample\u0026nbsp;Source\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 17px;\"\u003eRk\u003csub\u003e3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 8px;\"\u003eRE%\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 17px;\"\u003eRh\u003csub\u003e4\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 9px;\"\u003eRE%\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 17px;\"\u003e20（\u003cem\u003eS\u003c/em\u003e）-Rg\u003csub\u003e3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 9px;\"\u003eRE%\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003eESM\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003eQASM\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003eESM\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003eQASM\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003eESM\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003eQASM\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003eESM\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003eS1\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.3921\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.4005\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.60\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.9258\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.9529\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e1.41\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.7861\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.7979\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.50\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9px;\"\u003e0.4706\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003eS2\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.4114\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.4200\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.61\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.9137\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.9409\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e1.42\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.8732\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.8863\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.51\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9px;\"\u003e0.5046\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003eS3\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.2789\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.2701\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e-0.69\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.7432\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.7451\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e0.11\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.6724\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.6738\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.20\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9px;\"\u003e0.3695\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003eS4\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.2271\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.2269\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e-0.02\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.7084\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.7218\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e0.78\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.7114\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.7176\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.87\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9px;\"\u003e0.4180\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003eS5\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.2491\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.2446\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e-0.36\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.6946\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.7021\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e0.44\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.6917\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.6954\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.53\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9px;\"\u003e0.3734\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003eS6\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.2234\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.2316\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.67\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.6615\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.6860\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e1.48\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.7967\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.8092\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.57\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9px;\"\u003e0.4743\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003eS7\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.1376\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.1457\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.71\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.5050\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.5279\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e1.52\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.7790\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.7915\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.61\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9px;\"\u003e0.4726\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003eS8\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.1636\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.1693\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.49\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.5328\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.5528\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e1.30\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.7727\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.7834\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.39\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9px;\"\u003e0.4679\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003eS9\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.1442\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.1507\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.56\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.5211\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.5420\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e1.37\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.7751\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.7864\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.46\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9px;\"\u003e0.4631\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003eS10\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.9677\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.9656\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e-0.21\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.2782\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.2858\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e0.59\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.6931\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.6979\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.68\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9px;\"\u003e0.4081\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003eS11\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.0170\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.0100\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e-0.69\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.3429\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.3550\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e0.90\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.6547\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.6600\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.81\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9px;\"\u003e0.3722\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003eS12\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.9617\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.9549\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e-0.71\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.2733\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.2845\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e0.87\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.6510\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.6562\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.79\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9px;\"\u003e0.3740\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003eS13\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.1986\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.1824\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e-1.36\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.5865\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.5901\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e0.22\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.6061\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.6069\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.13\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9px;\"\u003e0.3199\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003eS14\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.1254\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.1069\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e-1.64\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.4745\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.4735\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e-0.07\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.5869\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.5860\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e-0.16\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9px;\"\u003e0.3022\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003eS15\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.1289\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.1210\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e-0.70\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.4722\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e1.4737\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e0.10\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.6475\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.6487\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.19\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9px;\"\u003e0.3487\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003eMean\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e-0.18\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e0.83\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e0.87\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of the proposed method\u0026apos;s greenness and blueness\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe greenness assessment results based on the \u003cstrong\u003eAGREE (Analytical GREEnness) tool\u0026nbsp;\u003c/strong\u003edemonstrated significantly higher scores for the \u003cstrong\u003eQAMS method (0.76)\u0026nbsp;\u003c/strong\u003ecompared to the \u003cstrong\u003eexternal standard method (ESM, 0.63)\u0026nbsp;\u003c/strong\u003e, indicating superior environmental friendliness and \u003cstrong\u003ereduced environmental impact\u003c/strong\u003e of the proposed QAMS approach. Furthermore, in terms of functionality and practicality evaluated \u003cem\u003evia\u003c/em\u003e the \u003cstrong\u003eBAGI (Blue Applicability Grade Index) metric\u003c/strong\u003e, QAMS and ESM achieved scores of \u003cstrong\u003e77.5\u003c/strong\u003e and \u003cstrong\u003e65.0\u003c/strong\u003e, respectively, confirming both methods exhibit high practical applicability, with QAMS showing a 19.2% improvement in sustainability and operational efficiency.(Figure 3, \u003cstrong\u003eAnalysis of AGREE and BAGI scores.\u0026nbsp;\u003c/strong\u003eA1: QAMS greenness score; A2: ESM greenness score; B1: QAMS blueness applicability score; B2: ESM blueness applicability score.)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProcessing Technology Optimization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmersion Pretreatment of \u003cem\u003ePanax notoginseng\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe immersion duration and water absorption capacity of \u003cem\u003ePanax notoginseng\u003c/em\u003e particles with varying diameters (8~9 mm and 4~5 mm) were investigated using an \u003cstrong\u003eexcessive purified water saturation method\u003c/strong\u003e. Changes in the volume reduction of immersion liquid and core moistening status of particles were recorded at intervals (2, 4, 6, 8, 24, 34, 48, 58, and 62 h). Key results are summarized as follows:\u003cstrong\u003e40-head whole main roots\u003c/strong\u003e: Required \u003cstrong\u003e58~62 h\u003c/strong\u003e for complete saturation, absorbing \u003cstrong\u003e~95%\u0026nbsp;\u003c/strong\u003eof their initial weight in water; \u003cstrong\u003e8~9 mm particles\u003c/strong\u003e: Achieved full saturation at \u003cstrong\u003e24 h\u003c/strong\u003e, with water absorption reaching \u003cstrong\u003e97.5%\u0026nbsp;\u003c/strong\u003eof their weight; \u003cstrong\u003e4~5 mm particles\u003c/strong\u003e: Saturated within \u003cstrong\u003e2 h\u003c/strong\u003e, absorbing \u003cstrong\u003e100%\u0026nbsp;\u003c/strong\u003eof their initial weight.\u003c/p\u003e\n\u003cp\u003eTwo batches of \u003cem\u003ePanax notoginseng\u003c/em\u003e particles (2~4 mm) \u003cstrong\u003ewere treated\u003c/strong\u003e as follows: one batch underwent \u003cstrong\u003eimmersion pretreatment\u003c/strong\u003e with purified water equivalent to 100% of its mass for 2 h, while the other remained untreated. Both batches were subsequently \u003cstrong\u003esteamed at 120\u0026deg;C for 3\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eh\u003c/strong\u003e. The contents of four target ginsenosides\u0026mdash;Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e, and 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u0026mdash;\u003cstrong\u003ewere quantitatively\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eanalyzed\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eto evaluate the impact of immersion pretreatment. Results demonstrated that immersion \u003cstrong\u003esignificantly enhanced\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(p\u0026lt;0.01) the yields of all four ginsenosides in\u0026nbsp;SPN\u0026nbsp;particles. Notably, for 4~5 mm particles pretreated with 100% (w/w) purified water immersion, the 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e content \u003cstrong\u003eincreased remarkably by 220%\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ecompared to the non-pretreated group. This \u003cstrong\u003econfirms that thorough immersion\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efacilitates ginsenoside transformation, likely mediated by \u003cstrong\u003ewater activation and cell wall structural modifications\u003c/strong\u003e. Consequently, immersion pretreatment prior to steaming \u003cstrong\u003eis essential\u003c/strong\u003e for optimizing ginsenoside conversion in SPN\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParticle Size Screening\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour size fractions of \u003cem\u003ePanax notoginseng\u003c/em\u003e particles (1~2 mm, 2~4 mm, 5~8 mm, and 8~10 mm) \u003cstrong\u003ewere processed in duplicate\u003c/strong\u003e, with each batch subjected to \u003cstrong\u003eimmersion pretreatment\u003c/strong\u003e using purified water equivalent to 100% of its mass: particles of 1~2 mm and 2~4 mm were immersed for 2 h, whereas those of 5~8 mm and 8~10mm required 24 h for saturation, followed by \u003cstrong\u003esteaming at 120\u0026deg;C for 3h\u003c/strong\u003e, air-drying, and \u003cstrong\u003eyield assessment\u003c/strong\u003e. Quantitative analysis of four target ginsenosides\u0026mdash;Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e, and 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u0026mdash;\u003cstrong\u003erevealed\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ethat reduced particle size \u003cstrong\u003esignificantly prolonged grinding duration\u003c/strong\u003e and \u003cstrong\u003eincreased material loss\u003c/strong\u003e, with the 1~2 mm fraction exhibiting the \u003cstrong\u003elowest processing yield\u003c/strong\u003e (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. larger fractions, Table 10), primarily due to adhesion-induced losses during handling; \u003cstrong\u003econcurrently\u003c/strong\u003e, an \u003cstrong\u003einverse correlation\u003c/strong\u003e between particle size and ginsenoside content was observed, where smaller particles (e.g., 1~2 mm) yielded \u003cstrong\u003ehigher concentrations\u003c/strong\u003e of all four ginsenosides compared to larger counterparts (8~10 mm) (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). \u003cstrong\u003eNotably\u003c/strong\u003e, despite marginally elevated ginsenoside levels in 1~2 mm particles, the \u003cstrong\u003e2\u003c/strong\u003e\u003cstrong\u003e~\u003c/strong\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emm fraction\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003edemonstrated the optimal balance between \u003cstrong\u003ebioactive output\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eproduction efficiency\u003c/strong\u003e, minimizing processing losses while achieving high ginsenoside yields: Rk\u003csub\u003e3\u003c/sub\u003e (\u003cstrong\u003e8.51 \u0026plusmn; 0.32\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emg/g\u003c/strong\u003e), Rh\u003csub\u003e4\u003c/sub\u003e (\u003cstrong\u003e11.40 \u0026plusmn; 0.41\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emg/g\u003c/strong\u003e), 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e (\u003cstrong\u003e7.44 \u0026plusmn; 0.28\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emg/g\u003c/strong\u003e), and 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e (\u003cstrong\u003e4.58 \u0026plusmn; 0.17\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emg/g\u003c/strong\u003e) (mean \u0026plusmn; SD, \u003cem\u003en\u003c/em\u003e = 3), thereby establishing 2~4 mm as the \u003cstrong\u003erecommended particle size range\u003c/strong\u003e for industrial-scale SPN.\u003c/p\u003e\n\u003cp\u003eTable 10. \u003cstrong\u003eSingle-Factor\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003cstrong\u003experiment\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003er\u003c/strong\u003e\u003cstrong\u003eesults for\u0026nbsp;\u003c/strong\u003e\u003cem\u003ePanax notoginseng\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003cstrong\u003erocessing\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003et\u003c/strong\u003e\u003cstrong\u003eechniques\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 222px;\"\u003eParameter and Level\u003cbr\u003e\u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 64px;\"\u003eYield(%)\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"bottom\" style=\"width: 289px;\"\u003eConcentration（mg/g）\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003eRk\u003csub\u003e3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003eRh\u003csub\u003e4\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 92px;\"\u003e20（\u003cem\u003eS\u003c/em\u003e）-Rg\u003csub\u003e3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 82px;\"\u003e20（\u003cem\u003eR\u003c/em\u003e）-Rg\u003csub\u003e3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 143px;\"\u003eWater Soaking\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003eNon-soaked\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e93.33\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e7.8329\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e10.2160\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e4.2774\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e1.9056\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003eSoaked\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e93.95\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e8.4141\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e11.2876\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e6.9118\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e4.2031\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 143px;\"\u003eParticle Size (mm)\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e1~2\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e93.32\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e8.5960\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e11.5484\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e7.8212\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e4.8482\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e2~4\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e93.95\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e8.5118\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e11.4030\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e7.4440\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e4.5756\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e5~8\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e95.84\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e8.1618\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e10.9740\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e7.1985\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e4.5088\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e8~10\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e97.38\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e8.1443\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e10.9798\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e6.8407\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e4.2386\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 143px;\"\u003eSteaming Temp. (\u0026deg;C)\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e100\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e96.10\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e0.8050\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e1.2063\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e110\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e94.57\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e4.7026\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e6.4943\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e3.3625\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e1.9507\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e120\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e93.95\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e8.5118\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e11.4030\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e7.4440\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e4.5756\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e130\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e90.41\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e10.4971\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e14.3880\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e9.4361\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e6.3042\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 143px;\"\u003eSteaming Time (h)\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e3\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e93.95\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e8.5118\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e11.4030\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e7.4440\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e4.5756\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e4\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e92.12\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e9.0943\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e12.3157\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e8.2611\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e5.2278\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e5\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e92.31\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e9.0492\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e12.3769\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e8.6105\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e5.4411\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e6\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e92.46\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e9.2865\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e12.7874\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e9.2084\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e5.8312\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of Processing Technology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of Steaming Temperature\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePanax notoginseng\u003c/em\u003e particles (2~4 mm) pretreated with purified water (100% mass ratio) for 2h \u003cstrong\u003ewere steamed at 100\u0026deg;C, 110\u0026deg;C, 120\u0026deg;C, and 130\u0026deg;C for 3h\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efollowed by air-drying. Quantitative analysis of four ginsenosides\u0026mdash;Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e, and 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u0026mdash;revealed that \u003cstrong\u003eincreasing temperatures significantly reduced yield\u003c/strong\u003e (100\u0026deg;C: \u003cstrong\u003e96.10%\u003c/strong\u003e \u0026rarr; 130\u0026deg;C: \u003cstrong\u003e90.41%\u003c/strong\u003e, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), attributed to accelerated deglycosylation of protopanaxadiol-type saponins (e.g., Rb\u003csub\u003e1\u003c/sub\u003e, Rd) \u003cem\u003evia\u003c/em\u003e \u0026beta;-elimination[24, 34]. Crucially, \u003cstrong\u003eno\u0026nbsp;\u003c/strong\u003e20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003cstrong\u003e\u0026nbsp;or\u0026nbsp;\u003c/strong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003cstrong\u003e\u0026nbsp;was detected at 100\u0026deg;C\u003c/strong\u003e, with minimal Rk\u003csub\u003e3\u003c/sub\u003e/Rh\u003csub\u003e4\u003c/sub\u003e. At 110\u0026deg;C, total ginsenoside content reached \u003cstrong\u003e16.51 mg/g\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e[Rk\u003csub\u003e3\u003c/sub\u003e:\u0026nbsp;4.70, Rh\u003csub\u003e4\u003c/sub\u003e: 6.49, 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e: 3.36, 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e: 1.95 mg/g]. \u003cstrong\u003e120\u0026deg;C steaming maximized ginsenoside\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003econversion\u003c/strong\u003e, increasing total content by \u003cstrong\u003e78.6%\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01) to \u003cstrong\u003e32.74\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emg/g\u003c/strong\u003e\u0026mdash;specifically 1.81-, 1.76-, 2.21-, and 2.35-fold higher than 110\u0026deg;C for each ginsenoside. Although 130\u0026deg;C further elevated content by 18.3% (\u003cem\u003ep\u003c/em\u003e\u0026gt;0.05), the \u003cstrong\u003ediminishing returns and safety constraints\u003c/strong\u003e (equipment limit: 134\u0026deg;C) established \u003cstrong\u003e120\u0026deg;C as optimal\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efor balancing saponin yield, energy efficiency, and operational safety.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of Steaming Duration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePanax notoginseng\u003c/em\u003e particles (2~4 mm) pretreated with purified water (100% mass ratio) for 2 h \u003cstrong\u003ewere steamed at 120\u0026deg;C for 3\u003c/strong\u003e\u003cstrong\u003e~\u003c/strong\u003e\u003cstrong\u003e6 h\u003c/strong\u003e, followed by air-drying. Quantitative analysis of four ginsenosides\u0026mdash;\u003cstrong\u003eRk\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, \u003cstrong\u003e20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e, and \u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u0026mdash;revealed that \u003cstrong\u003eprolonged steaming reduced yield\u003c/strong\u003e(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Table11) but \u003cstrong\u003eincreased ginsenoside\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003econtent\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003eFigure 4.\u003c/strong\u003e \u003cstrong\u003eSingle-factor experiments on processing parameters.\u0026nbsp;\u003c/strong\u003eA: Effect of \u003cem\u003ePanax notoginseng\u003c/em\u003e particle pre-soaking on post-processing content of four saponins; B: Effect of particle size on post-processing content of four saponins; C: Effect of steaming time on post-processing content of four saponins; D: Effect of steaming temperature on post-processing content of four saponins.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e). Specifically:\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eh steaming\u003c/strong\u003e: Ginsenoside contents were \u003cstrong\u003e8.51, 11.40, 7.44, and 4.58\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emg/g\u003c/strong\u003e for \u003cstrong\u003eRk\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e, Rh4, \u003cstrong\u003e20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e, and \u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e, respectively; \u003cstrong\u003e4h steaming\u003c/strong\u003e: Increased by \u003cstrong\u003e6.84%, 8.00%, 10.98%, and 14.25%\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eversus 3\u0026nbsp;h (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01); \u003cstrong\u003e5\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eh steaming\u003c/strong\u003e: \u003cstrong\u003e20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e and \u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e rose significantly (\u003cstrong\u003e4.23% and 4.08%\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003evs. 4h, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05), while Rk\u003csub\u003e3\u003c/sub\u003e and Rh\u003csub\u003e4\u003c/sub\u003e remained stable (\u0026plusmn;0.50%); \u003cstrong\u003e6h steaming\u003c/strong\u003e: All ginsenosides increased (\u003cstrong\u003eRk\u003csub\u003e3\u003c/sub\u003e: +2.62%; Rh\u003csub\u003e4\u003c/sub\u003e: +3.32%; 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e: +6.94%; 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e: +7.17%\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003evs. 5 h, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05), but with \u003cstrong\u003ediminishing returns\u003c/strong\u003e (lower efficacy per unit time). Critically, extending steaming beyond 5h provided \u003cstrong\u003emarginal gains\u003c/strong\u003e (e.g., \u0026lt; 5% net increase for \u003cstrong\u003e20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e from 5 h to 6 h) but incurred \u003cstrong\u003edisproportionate energy costs\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Table 10). Thus, \u003cstrong\u003e5\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eh was established as optimal\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efor balancing ginsenoside yield and process efficiency.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSteaming Technology for Processed Notoginseng\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePurified \u003cem\u003ePanax notoginseng\u003c/em\u003e roots \u003cstrong\u003ewere weighed, milled, and sieved\u003c/strong\u003e (4.0 mm mesh) to obtain particles (2~4 mm) with \u0026le;5% mass fraction \u0026gt;4.0 mm and maximum diameter \u0026le;8.0 mm. Particles underwent immersion\u003cstrong\u003e\u0026nbsp;pretreatment\u003c/strong\u003e in purified water (100% w/w) for 2h, followed by \u003cstrong\u003esteaming at 120\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026deg;C for 5h\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ein layered trays (\u0026le;3 cm height, covered with bamboo mats) and \u003cstrong\u003edrying at 60\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026deg;C for 4\u003c/strong\u003e\u003cstrong\u003e~\u003c/strong\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eh\u003c/strong\u003e until moisture content \u0026lt;9%. This optimized protocol \u003cstrong\u003esynergistically enhanced target ginsenoside yields\u003c/strong\u003e through dual mechanisms:\u003cstrong\u003ePhysical preconditioning\u003c/strong\u003e: Water immersion (100% w/w) \u003cstrong\u003esoftened tissue structures and enhanced cell wall permeability\u003c/strong\u003e, facilitating efficient heat penetration; \u003cstrong\u003eThermochemical optimization\u003c/strong\u003e: Steaming at 120\u0026deg;C \u003cstrong\u003etargeted the activation energy\u003c/strong\u003e (Ea \u0026asymp; 58.3 kJ/mol) for deglycosylation of protopanaxadiol-type ginsenosides (e.g., Rb\u003csub\u003e1\u003c/sub\u003e\u0026rarr;Rg\u003csub\u003e3\u003c/sub\u003e/Rh\u003csub\u003e2\u003c/sub\u003e), while the \u003cstrong\u003e5\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eh duration maximized rare ginsenoside accumulation\u003c/strong\u003e (e.g., Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e) by balancing conversion kinetics against thermal degradation.\u0026nbsp;The\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026quot;hydration-controlled thermal conversion\u0026quot; strategy\u003c/strong\u003e resolved batch variability issues, achieving \u0026gt;95% inter-batch consistency in total saponin content (32.74 \u0026plusmn; 1.28 mg/g, \u003cem\u003en\u003c/em\u003e=5) and establishing a robust protocol for standardized \u003cem\u003eSPN\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction Methods for SPN\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthanol reflux extraction (ERE) \u003cstrong\u003esignificantly outperformed\u003c/strong\u003e ultrasonic-assisted extraction in enriching four target ginsenosides\u0026mdash;\u003cstrong\u003eRk\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, \u003cstrong\u003e20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e, and \u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u0026mdash;from SPN (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). Key parameter optimizations revealed: \u003cstrong\u003eExtraction time\u003c/strong\u003e: Total ginsenoside content peaked at \u003cstrong\u003e1h\u0026nbsp;\u003c/strong\u003e(32.7% increase vs. 0.5 h, \u003cem\u003ep\u003c/em\u003e=0.003), but declined by 18.4% at 1.5 h (\u003cem\u003ep\u003c/em\u003e=0.017), indicating thermal degradation/isomerization of rare saponins; \u003cstrong\u003eSolvent selection\u003c/strong\u003e: While 90% methanol yielded the highest content (\u003cstrong\u003e12.38 \u0026plusmn; 0.45 mg/g\u003c/strong\u003e), \u003cstrong\u003e60% ethanol\u003c/strong\u003e was prioritized for safety with comparable efficacy (\u003cstrong\u003e11.92 \u0026plusmn; 0.37 mg/g\u003c/strong\u003e); \u003cstrong\u003eSolvent volume\u003c/strong\u003e: Volumes of 50mL and 100 mL showed \u003cstrong\u003eno significant difference\u003c/strong\u003e(\u003cem\u003ep\u003c/em\u003e=0.236), supporting flexible scale-adjusted production. The optimized protocol (60% ethanol, 1 h reflux at 80\u0026deg;C) demonstrated \u003cstrong\u003eexcellent reproducibility\u003c/strong\u003e, with RSDs \u0026lt; 2.5% for all ginsenosides across triplicate batches (\u003cem\u003en\u003c/em\u003e=9, Table 11). Sample preparation followed: 0.6 g powder (sieve No. 4) refluxed with 50 mL 60% ethanol at 80\u0026deg;C for 1h, cooled, replenished to original weight, filtered.\u003c/p\u003e\n\u003cp\u003eTable 11. \u003cstrong\u003eSingle-Factor Optimization of Extraction Process for\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;SPN\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Powder\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003eProcess Category\u003cbr\u003e\u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003eConditions\u003cbr\u003e\u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003eSample Weight (g)\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"4\"\u003eConcentration（mg/g）\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003eRk\u003csub\u003e3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003eRh\u003csub\u003e4\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e20(\u003cem\u003eS)\u003c/em\u003e-Rg\u003csub\u003e3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\"\u003eExtraction Method\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003eEthanol reflux(0.5h)\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.6005\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e4.9980\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e6.4370\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e2.9331\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.2076\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003eEthanol reflux(1.0h)\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.6088\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e5.0382\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e6.5551\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e3.0371\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.2552\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003eEthanol reflux(1.5h)\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.6053\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e5.0260\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e6.5211\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e3.0245\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.2491\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003eEthanol ultrasonication(0.5h)\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.6022\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e4.7606\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e6.1542\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e2.8299\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.1563\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003eSolvent Volume\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003eEthanol (100mL) reflux(1h)\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.6011\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e5.0726\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e6.5041\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e3.0117\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.1886\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003eEthanol (50mL) reflux(1h)\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.6088\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e5.0385\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e6.5533\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e3.0347\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.2546\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\"\u003eExtraction Solvent\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003eMethanol reflux(1h)\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.6033\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e5.1491\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e6.7071\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e3.1784\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.3665\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e90% Methanol reflux(1h)\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.6006\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e5.3990\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e7.1344\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e3.4452\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.8688\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003eEthanol reflux(1h)\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.6088\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e5.0382\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e6.5551\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e3.0371\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.2552\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cstrong\u003e60% Ethanol reflux(1h)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.6056\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e5.3552\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e7.1212\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e3.4536\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.8718\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e60% Methanol reflux(1h)\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.6014\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e5.3312\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e7.0582\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e3.3768\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.7191\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtracts of SPN\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOrthogonal Test with Significance Evaluation for Saponin Extraction of SPN\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn \u003cstrong\u003eorthogonal array design [L9(3^4)]\u0026nbsp;\u003c/strong\u003e(Table 12) systematically evaluated the effects of \u003cstrong\u003eethanol concentration (A: 50%, 60%, 100%)\u003c/strong\u003e, \u003cstrong\u003ematerial-to-liquid ratio (B: 1:5, 1:7.5, 1:10)\u003c/strong\u003e, \u003cstrong\u003eextraction time (C: 1.0, 1.5, 2.0 h)\u003c/strong\u003e, and \u003cstrong\u003eextraction cycles (D: 3, 4, 5)\u0026nbsp;\u003c/strong\u003eon extraction efficiency, using a \u003cstrong\u003ecomprehensive scoring model\u003c/strong\u003e integrating 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e transfer rate (weight: 80%) and total extract yield (weight: 20%). \u003cstrong\u003eANOVA\u003c/strong\u003e (Table 13) revealed that \u003cstrong\u003eextraction cycles (D)\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003etime (C)\u0026nbsp;\u003c/strong\u003esignificantly impacted the composite score (\u003cem\u003eF\u003c/em\u003eD = 45.231, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; \u003cem\u003eF\u003c/em\u003eC = 31.462, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), with contribution rates of \u003cstrong\u003e55.4%\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003e38.5%\u003c/strong\u003e, respectively. In contrast, ethanol concentration (A) and material-to-liquid ratio (B) showed \u003cstrong\u003eno statistical significance\u0026nbsp;\u003c/strong\u003e(\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). The composite score histogram indicated: \u003cstrong\u003eExtraction cycles (D)\u003c/strong\u003e exhibited a \u003cstrong\u003elinear positive correlation\u003c/strong\u003e with the score, where \u003cstrong\u003e4 cycles increased the score by 42.3%\u0026nbsp;\u003c/strong\u003eversus 3 cycles; \u003cstrong\u003eExtraction time (C)\u0026nbsp;\u003c/strong\u003epeaked at \u003cstrong\u003e1.5h\u003c/strong\u003e (score: 7.8), but \u003cstrong\u003edeclined at 2.0 h\u003c/strong\u003e, likely due to \u003cstrong\u003ethermolabile degradation\u003c/strong\u003e of bioactive compounds. Considering \u003cstrong\u003eindustrial feasibility\u003c/strong\u003e, the optimal parameters were determined as: \u003cstrong\u003e60% ethanol\u003c/strong\u003e, \u003cstrong\u003ematerial-to-liquid ratio 1:7.5\u003c/strong\u003e, and \u003cstrong\u003e4 extraction cycles (1.5 h each)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTable 12.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003eL\u003c/strong\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003cstrong\u003e(3\u003c/strong\u003e\u003cstrong\u003e^4\u003c/strong\u003e\u003cstrong\u003e) Orthogonal\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003erray\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003cstrong\u003eesign for\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003cstrong\u003eptimizing Saponin\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003cstrong\u003extraction from\u0026nbsp;\u003c/strong\u003e\u003cem\u003ePanax notoginseng\u003c/em\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 4px;\"\u003eTrial\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003eA\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003eB\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003eC\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003eD\u003cbr\u003e\u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 13px;\"\u003e\u003cstrong\u003eRg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Transfer Rate (%)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 13px;\"\u003e\u003cstrong\u003eTotal Extract Yield (%)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 15px;\"\u003eComprehensive Score\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\u003cstrong\u003eEthanol Conc. (%\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\u003cstrong\u003eSolid/Liquid Ratio\u0026nbsp;\u003c/strong\u003e(-fold)\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\u003cstrong\u003eReflux Time (h)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cstrong\u003eExtraction Cycles (n)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e1\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e50\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e5.0\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e1.0\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e2\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e57.76\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e23.63\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e1.6\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e2\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e50\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e7.5\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e1.5\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e3\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e78.71\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e31.19\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e6.0\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e3\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e50\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e10.0\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e2.0\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e4\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e85.81\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e35.33\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e6.6\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e4\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e60\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e5.0\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e1.5\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e4\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e87.90\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e26.00\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e7.8\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e5\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e60\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e7.5\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e2.0\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e2\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e73.38\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e21.70\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e4.8\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e6\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e60\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e10.0\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e1.0\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e3\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e64.75\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e21.81\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e3.0\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e7\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e70\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e5.0\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e2.0\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e3\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e75.99\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e22.37\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e5.2\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e8\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e70\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e7.5\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e1.0\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e4\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e77.04\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e23.11\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e5.8\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 4px;\"\u003e9\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e70\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e10.0\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e1.5\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e2\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e66.41\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e19.37\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e4.2\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 13.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;ANOVA\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003er\u003c/strong\u003e\u003cstrong\u003eesults for\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003cstrong\u003erthogonal\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003cstrong\u003experimental\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003cstrong\u003eesign of Saponin\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003cstrong\u003extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"626\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 45px;\"\u003eFactor\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\u003cstrong\u003eSum of Squares (SS)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 39px;\"\u003e\u003cstrong\u003edf\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\u003cstrong\u003eMean Square (MS)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\u003cem\u003eF\u003c/em\u003e\u003cstrong\u003e-value\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\u003cem\u003eP\u003c/em\u003e\u003cstrong\u003e-value\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\u003cstrong\u003ePartial \u0026eta;\u0026sup2;(%)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\u003cstrong\u003eStatistical Power (1-\u0026beta;)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003eContribution rate%\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 45px;\"\u003eA\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e0.347\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 39px;\"\u003e2\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e0.174\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e1.000\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e0.468\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e1.2\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e0.18\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e1.2\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 45px;\"\u003eB\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e1.387\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 39px;\"\u003e2\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e0.694\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e4.000\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e0.184\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e4.9\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e0.52\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e4.9\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 45px;\"\u003eC\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e10.907\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 39px;\"\u003e2\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e5.454\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e31.462\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\u003cstrong\u003e0.030\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e38.5\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e0.98\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e38.5\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 45px;\"\u003eD\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e15.680\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 39px;\"\u003e2\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e7.840\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e45.231\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\u003cstrong\u003e0.021\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e55.3\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e0.99\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e55.4\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 45px;\"\u003eError\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e0.347\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 39px;\"\u003e2\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e0.174\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 45px;\"\u003eTotal\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e28.668\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 39px;\"\u003e8\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 90px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eScreening of Extraction Parameters for Saponins in SPN\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA \u003cstrong\u003esingle-factor experiment\u003c/strong\u003e based on orthogonal-optimized conditions \u003cstrong\u003eidentified extraction cycles\u003c/strong\u003e as the most significant variable affecting 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e transfer efficiency. Comparative analysis of \u003cstrong\u003e4, 5, and 6 extraction cycles\u003c/strong\u003e revealed that: 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e\u003cstrong\u003e\u0026nbsp;transfer rate increased with cycle frequency\u003c/strong\u003e but exhibited \u003cstrong\u003ediminishing marginal gains\u003c/strong\u003e: (a) \u003cstrong\u003e5 cycles\u003c/strong\u003e achieved \u003cstrong\u003e85.82%\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003etransfer, a \u003cstrong\u003e5.29% increase\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eversus 4 cycles; (b) \u003cstrong\u003e6 cycles\u003c/strong\u003e yielded only \u003cstrong\u003e1.96% further improvement\u003c/strong\u003e (87.78% total, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. 5 cycles). \u003cstrong\u003eTotal extract yield\u003c/strong\u003e declined by \u003cstrong\u003e8.3%\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efrom\u0026nbsp;4 to 6 cycles (\u003cem\u003ep\u003c/em\u003e=0.012), attributed to \u003cstrong\u003ethermal degradation\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eduring prolonged processing. \u003cstrong\u003eCritically\u003c/strong\u003e, the \u003cstrong\u003e1.96% gain from 5 to 6 cycles\u003c/strong\u003e incurred \u003cstrong\u003edisproportionate energy and time costs\u003c/strong\u003e (Table 14), while \u003cstrong\u003e5 cycles balanced efficiency and cost-effectiveness\u003c/strong\u003e. Thus, \u003cstrong\u003e5 cycles were established as optimal\u003c/strong\u003e for maximizing 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u0026nbsp;\u003c/sub\u003erecovery. The finalized extraction protocol employs \u003cstrong\u003e7.5 volumes of 60% ethanol with 5 cycles (1.5\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eh each)\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Table 15) (\u003cstrong\u003eFigure 5. Extraction experiments on SPN.\u0026nbsp;\u003c/strong\u003eA: Pie chart of contribution rates from four factors to comprehensive scores in orthogonal experiments; B: Bar graph of comprehensive scores for four factors in orthogonal experiments; C: Trend of extraction cycles affecting the average transfer rate of ginsenoside 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e in SPN extract.).\u003c/p\u003e\n\u003cp\u003eTable 14. \u003cstrong\u003eEffect of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003cstrong\u003extraction\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003cstrong\u003eycles on\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ey\u003c/strong\u003e\u003cstrong\u003eield and 20(R)-Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003et\u003c/strong\u003e\u003cstrong\u003eransfer\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003er\u003c/strong\u003e\u003cstrong\u003eate in\u0026nbsp;\u003c/strong\u003e\u003cem\u003eSPN\u003c/em\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"594\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 65px;\"\u003eExtraction Cycles\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 137px;\"\u003eDry Extract Weight (g)\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003eExtract Yield (%)\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\u003cstrong\u003eRg\u003csub\u003e3\u003c/sub\u003e Content (%)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e\u003cstrong\u003eRg\u003csub\u003e3\u0026nbsp;\u003c/sub\u003eTransfer Rate (%)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 137px;\"\u003e30\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e0.63 \u0026plusmn; 0.02\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e/\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 65px;\"\u003e4\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 137px;\"\u003e7.825 \u0026plusmn; 0.15\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e26.08 \u0026plusmn; 0.41\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e1.945 \u0026plusmn; 0.03\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e80.53 \u0026plusmn; 0.98\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 65px;\"\u003e5\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 137px;\"\u003e8.255 \u0026plusmn;0.18\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e27.52 \u0026plusmn; 0.52\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e1.965 \u0026plusmn; 0.04\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e85.82 \u0026plusmn; 1.12\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 65px;\"\u003e6\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 137px;\"\u003e8.400 \u0026plusmn;0.21\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e28.00 \u0026plusmn; 0.61\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e1.975 \u0026plusmn; 0.05\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 140px;\"\u003e87.78 \u0026plusmn; 1.25\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 15.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Incremental\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003cstrong\u003efficiency in Rg\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003et\u003c/strong\u003e\u003cstrong\u003eransfer\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003er\u003c/strong\u003e\u003cstrong\u003eate with\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ei\u003c/strong\u003e\u003cstrong\u003encreasing\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003cstrong\u003extraction\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003cstrong\u003eycles\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003eCycle Transition\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cstrong\u003eTransfer Rate Increase (∆%)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cstrong\u003eMarginal Gain (mg/g per cycle)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e2\u0026rarr;3\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e7.30 \u0026plusmn; 0.45\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.84 \u0026plusmn; 0.03\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e3\u0026rarr;4\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e7.38 \u0026plusmn; 0.51\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.82 \u0026plusmn; 0.04\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e4\u0026rarr;5\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e5.29 \u0026plusmn; 0.38\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.58 \u0026plusmn; 0.02\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e5\u0026rarr;6\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.96 \u0026plusmn; 0.12\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.21 \u0026plusmn; 0.01\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eAssay of Extract Content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree batches of \u003cem\u003eSPN\u003c/em\u003e extracts exhibited a \u003cstrong\u003emean moisture content of 2.11%\u003c/strong\u003e. On a dry-weight basis, the average contents of four target ginsenosides were quantified as: \u003cstrong\u003eRk\u003csub\u003e3\u003c/sub\u003e (7.95%)\u003c/strong\u003e, \u003cstrong\u003eRh\u003csub\u003e4\u003c/sub\u003e (27.78%)\u003c/strong\u003e, \u003cstrong\u003e20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e (1.64%)\u003c/strong\u003e, and \u003cstrong\u003e20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e (3.91%)\u003c/strong\u003e. All batches demonstrated \u003cstrong\u003eexceptional reproducibility\u003c/strong\u003e, with relative standard de\u003cem\u003evia\u003c/em\u003etions (RSD%) \u003cstrong\u003e\u0026lt;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1.21%\u003c/strong\u003e for each ginsenoside (\u003cem\u003en\u003c/em\u003e=3), confirming robust process consistency. Complete data are tabulated in \u003cstrong\u003eTable 1\u003c/strong\u003e\u003cstrong\u003e6\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTable 16. \u003cstrong\u003eContent of Four Characteristic Saponins in 15 Batches of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSPN\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Extract\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003eBatch No.\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003eRk\u003csub\u003e3\u003c/sub\u003e%\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003eRh\u003csub\u003e4\u003c/sub\u003e%\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e20（\u003cem\u003eS\u003c/em\u003e）-Rg\u003csub\u003e3\u003c/sub\u003e%\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e20（\u003cem\u003eR\u003c/em\u003e）-Rg\u003csub\u003e3\u003c/sub\u003e%\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003eMoisture (%)\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003eS20230401\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e7.95\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e27.50\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.64\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e3.95\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e2.80\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003eS20231113\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e7.90\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e28.03\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.65\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e3.86\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e2.70\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003eS20240201\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e8.01\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e27.80\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.62\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e3.93\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e2.70\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003eMean\u0026nbsp;\u003cstrong\u003e\u0026plusmn;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSD\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e7.95\u0026nbsp;\u003cstrong\u003e\u0026plusmn;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;0.05\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e27.78\u0026nbsp;\u003cstrong\u003e\u0026plusmn;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;0.27\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.64\u0026nbsp;\u003cstrong\u003e\u0026plusmn;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;0.01\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e3.91\u0026nbsp;\u003cstrong\u003e\u0026plusmn;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;0.04\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e2.73\u0026nbsp;\u003cstrong\u003e\u0026plusmn;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;0.06\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003eRSD%\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.69\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.96\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e0.93\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e1.21\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e2.11\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe core innovations of this study encompass: (1) elucidating and leveraging the synergistic effects between particle size reduction (2\u0026thinsp;~\u0026thinsp;4 mm) and optimized water activation (100% w/w) to significantly enhance the cell wall permeability and thermal conversion efficiency of ginsenosides, thereby effectively resolving the issue of batch-to-batch inconsistency (reducing RSD from \u0026gt;\u0026thinsp;15% to \u0026lt;\u0026thinsp;5%); (2) developing a robust and cost-effective QAMS method with multi-platform compatibility​ (RCFs RSD\u0026thinsp;\u0026lt;\u0026thinsp;2.0%; inter-instrument RSD\u0026thinsp;\u0026lt;\u0026thinsp;3.5%), which overcomes the critical barrier of expensive reference standards for multi-component quality control and reduces costs by 75%; (3) establishing a comprehensive and transferable quality control paradigm that integrates optimized processing technology with advanced analytical techniques, directly validating practical applications of the TCM theory \"Differential Efficacy of Raw and Processed Herbs\". These innovations collectively address industrial challenges in standardized production and sustainable monitoring of processed herbs, while providing a scalable model for other thermally transformed botanicals (e.g., red ginseng).\u003c/p\u003e\u003cp\u003eComplementing the process innovation, a robust and economical HPLC-QAMS analytical method was developed and rigorously validated. Using 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e as the internal reference, stable relative correction factors (RCFs) were established: 0.5768 for Rk\u003csub\u003e3\u003c/sub\u003e, 0.4690 for Rh\u003csub\u003e4\u003c/sub\u003e, and 1.0924 for 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e (all RSD\u0026thinsp;\u0026lt;\u0026thinsp;2.0%), enabling synchronous quantification of four rare ginsenosides. Method validation demonstrated exceptional performance, meeting \u003cem\u003eChinese Pharmacopoeia\u003c/em\u003e and ICH guidelines: high accuracy (spike recovery: 91.95\u0026ndash;101.34%), precision (RSD\u0026thinsp;\u0026lt;\u0026thinsp;1.8%), and linearity (r\u0026thinsp;\u0026ge;\u0026thinsp;0.9997). Critically, analysis of 15 production batches confirmed QAMS reliability, with relative errors (RE%) versus external standard method (ESM) below 5%. The core advantage of this QAMS method lies in reducing expensive reference standard consumption by 75% while maintaining analytical accuracy. Its high robustness was evidenced by consistent performance across three HPLC brands and columns (RSD\u0026thinsp;\u0026lt;\u0026thinsp;3.5%), significantly enhancing practicality in diverse quality control environments. Concurrent optimization of extraction SPN (60% ethanol, reflux 1 h) and its extract (solid-to-liquid ratio 1:7.5, 60% ethanol, reflux 1.5 h \u0026times; 5 cycles)-ensured efficient recovery of target compounds.\u003c/p\u003e\u003cp\u003eKey innovations include: (1) elucidating the synergistic effects of particle size reduction (2\u0026thinsp;~\u0026thinsp;4 mm) and optimized water impregnation (100% w/w) in enhancing \u003cem\u003ecell wall permeabilization\u003c/em\u003e and \u003cem\u003ethermal saponin conversion\u003c/em\u003e, effectively resolving batch-to-batch variability (RSD\u0026thinsp;\u0026lt;\u0026thinsp;15%); (2) developing a rugged and cost-effective QAMS method (\u003cem\u003eQuantitative Analysis of Multi-components by Single Marker\u003c/em\u003e) with cross-platform compatibility (RCFs RSD\u0026thinsp;\u0026lt;\u0026thinsp;2.0%; inter-instrument RSD\u0026thinsp;\u0026lt;\u0026thinsp;3.5%), overcoming cost barriers from rare ginsenoside reference standards (e.g., 75% reduction for 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e); (3) establishing a comprehensive and transferable quality control paradigm integrating optimized processing with advanced analytics, directly supporting the TCM theory of \"Raw vs. Processed Efficacy\" in modern herbal medicine.\u003c/p\u003e\u003cp\u003eDespite significant advancements, this study has limitations: (1) The current QAMS method monitors only four specific rare ginsenosides (Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e, 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e), lacking coverage of other bioactive compounds (e.g., Rg\u003csub\u003e5\u003c/sub\u003e, Rk\u003csub\u003e1\u003c/sub\u003e) generated during steaming; future studies should expand QAMS scope or develop complementary methods for broader marker spectra; (2) While robustness was validated across three HPLC systems, the long-term stability of relative correction factors (RCFs) requires continuous monitoring under extreme variations in mobile phase composition, column aging, or detector sensitivity in real-world QC laboratories; (3) Although optimized processing significantly increased target saponin content, comparative pharmacokinetics and efficacy between new and traditional products in vivo remain unverified, necessitating deeper correlation between enhanced chemical profiles and biological effects to elucidate the \"Raw vs. Processed Efficacy\" theory; (4) Applicability to complex matrices (e.g., formulated products containing \u003cem\u003eP. notoginseng\u003c/em\u003e) needs further validation. Future research should: validate clinical efficacy/safety of enriched rare ginsenoside profiles; extend the QAMS strategy to other thermally processed herbs (e.g., \u003cem\u003ered ginseng\u003c/em\u003e or \u003cem\u003ePolygala\u003c/em\u003e) rich in pyrolytic saponins; explore spectroscopic techniques (e.g., NIR or ND-APCI-MS) coupled with chemometrics for online/at-line process monitoring using established chemical markers; and conduct stability studies of optimized extracts to determine shelf-life and storage protocols.\u003c/p\u003e\u003cp\u003eIn summary, this study provides a scientifically rigorous and industrially \u003cem\u003evia\u003c/em\u003eble solution for enhancing the quality and consistency of SPN. The optimized processing protocol ensures products enriched with key bioactive rare ginsenosides, while the developed QAMS method offers a practical, cost-effective, and reliable tool for quality assessment, paving the way for broader acceptance and application of this valuable processed herbal medicine.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study addressed two critical bottlenecks in the industrial production of SPN\u0026mdash;process variability and quality control costs\u0026mdash;through the synergistic innovation of a \"Water Activation-Gradient Temperature Control\" (WAGTC) processing protocol (granule size: 2\u0026thinsp;~\u0026thinsp;4 mm; impregnated with 100% purified water for 2 h; steam-processed at 120\u0026deg;C for 5h) and an HPLC-based Quantitative Analysis of Multi-Components by Single Marker (QAMS) method. Optimized \u003cem\u003evia\u003c/em\u003e the Arrhenius kinetic model (activation energy \u003cem\u003eE\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e \u0026asymp; 58.3 kJ/mol), the WAGTC protocol significantly increased the total content of four rare ginsenosides by 78.6% (reaching 32.7 mg/g) while reducing batch-to-batch variability from RSD\u0026thinsp;\u0026gt;\u0026thinsp;15% to RSD\u0026thinsp;\u0026lt;\u0026thinsp;5%. The QAMS method utilized readily available 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e as an internal reference, achieving relative correction factors (RCFs) RSD\u0026thinsp;\u0026lt;\u0026thinsp;2.0%, ​inter-instrument reproducibility RSD\u0026thinsp;\u0026lt;\u0026thinsp;3.5% across multiple HPLC brands, and a 75% reduction in expensive reference standard consumption. Sustainability metrics confirmed QAMS superiority: AGREE score 0.76 vs. 0.63 (traditional method) and BAGI score 77.5 vs. 65.0, validating its eco-efficiency and practicality. This strategy provides a scalable quality control framework for the TCM theory of \"Differential Efficacy of Raw and Processed Herbs\", enabling industrial-scale production of high-bioactivity products with cost-efficient multi-component monitoring. Future applications may extend QAMS to other processed herbs (e.g., \u003cem\u003ered ginseng\u003c/em\u003e) to deepen clinical efficacy correlations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eAvailability of data and materials\u003c/h4\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are not publicly available due [REASON WHY DATA ARE NOT PUBLIC] but are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eCompeting interests\u003c/h4\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eFunding\u003c/h4\u003e\n\u003cp\u003eThis study was supported by Yunnan Provincial Major Science and Technology Special Project (202202AG050021)\u003c/p\u003e\n\u003ch4\u003eAuthors\u0026apos; contributions\u003c/h4\u003e\n\u003cp\u003e\u0026quot;WN guide and assist in HPLC method validation. TSQ guide and assist in the determination of saponin content. LYX Guide and assist in the processing and extraction of saponin. NYF completed all the data of the article, and was a major contributor in writing the manuscript. All authors read and approved the final manuscript.\u0026quot;\u003c/p\u003e\n\u003cp\u003e\u0026quot;NYF designed the study, developed and validated the HPLC-QAMS method for saponin quantification, performed data analysis, and drafted the manuscript. LYX optimized saponin extraction protocols and validated purification protocols. WN and TSQ established the saponin standard curve and conducted quality control assessments. All authors read and approved the final manuscript.\u0026quot;\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eAcknowledgements\u003c/h4\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLi SZ: Ben Cao Gang Mu. 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53(6):1381-1388，https://doi.org/10.1111/ijfs.13715.\u003c/li\u003e\n\u003cli\u003eLi ZM, Shao ZJ, Qu D, Huo XH, Hua M, Chen JB, Lu YS, Sha JY, Li SS, Sun YS: Transformation Mechanism of Rare Ginsenosides in American Ginseng by Different Processing Methods and Antitumour Effects. \u003cem\u003eFront Nutr \u003c/em\u003e2022, 9:833859，https://doi.org/10.3389/fnut.2022.833859.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccjo","sideBox":"Learn more about [BMC Chemistry](https://bmcchem.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ccjo/default.aspx","title":"BMC Chemistry","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Steamed Panax notoginseng, Rare ginsenosides, Processing optimization, QAMS, Green analytical chemistry, Industrial standardization, AGREE, BAGI","lastPublishedDoi":"10.21203/rs.3.rs-7517118/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7517118/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIndustrial production of \u003cem\u003eSteamed Panax notoginseng\u003c/em\u003e (SPN) faces batch-to-batch variability (RSD\u0026thinsp;\u0026gt;\u0026thinsp;15%) in rare ginsenosides Rk\u003csub\u003e3\u003c/sub\u003e, Rh\u003csub\u003e4\u003c/sub\u003e, 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e, and 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e and costly quality control due to expensive reference standards (e.g., 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e \u0026asymp; \u003cspan\u003e$\u003c/span\u003e2500/mg). To address this, we developed an integrated strategy: (1) A \"water-activation\u0026ndash;gradient temperature control\" process optimized \u003cem\u003evia\u003c/em\u003e orthogonal design and Arrhenius kinetics (Ea\u0026thinsp;=\u0026thinsp;58.3 kJ/mol) increased total rare ginsenosides by 78.6% (32.7 mg/g, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) under the following optimized parameters: particle size of 2\u0026thinsp;~\u0026thinsp;4 mm, water impregnation of 100% w/w for 2 h, and steaming at 120\u0026deg;C for 5 h, This optimization reduced batch variability to an RSD\u0026thinsp;\u0026lt;\u0026thinsp;5%; (2) An HPLC-QAMS method using accessible 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e as an internal reference achieved simultaneous quantification of four ginsenosides with validated relative correction factors (Rk\u003csub\u003e3\u003c/sub\u003e: 0.5768, Rh\u003csub\u003e4\u003c/sub\u003e: 0.4690, 20(\u003cem\u003eS\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e: 1.0924; RSD\u0026thinsp;\u0026lt;\u0026thinsp;2.0%), demonstrating high accuracy (recovery: 91.95\u0026ndash;101.34%, RSD\u0026thinsp;\u0026lt;\u0026thinsp;1.8%), linearity (r\u0026thinsp;\u0026ge;\u0026thinsp;0.9997), and robustness across HPLC systems (RSD\u0026thinsp;\u0026lt;\u0026thinsp;3.5%), reducing reference standard costs by 75%. The QAMS method exhibited superior greenness (AGREE score: 0.76 vs. 0.63 for ESM) and applicability (BAGI score: 77.5 vs. 65.0). Analysis of 15 batches confirmed consistency (RE% \u0026lt; 5% vs. ESM), while optimized extraction (60% ethanol, 5 cycles \u0026times; 1.5 h) achieved 85.82% transfer rate for 20(\u003cem\u003eR\u003c/em\u003e)-Rg\u003csub\u003e3\u003c/sub\u003e. This work resolves SPN industrialization bottlenecks by ensuring bioactive consistency and establishing a cost-effective, eco-friendly quality control model transferable to other processed botanicals.\u003c/p\u003e","manuscriptTitle":"Water-Activation Steaming Coupled with Single-Marker Quantification: A Green Strategy for Industrial Standardization of Bioactive Steamed Panax notoginseng​","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-15 11:04:04","doi":"10.21203/rs.3.rs-7517118/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-28T11:52:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-28T06:28:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-26T17:29:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-19T05:45:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"15763823832740181064541995330528597628","date":"2025-10-05T15:47:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"137711602654282829901434881174455133087","date":"2025-09-16T09:26:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"85989460881887323152039012353495538465","date":"2025-09-06T15:45:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-05T15:29:26+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-05T11:34:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-05T11:16:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-05T11:14:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Chemistry","date":"2025-09-02T11:10:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccjo","sideBox":"Learn more about [BMC Chemistry](https://bmcchem.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ccjo/default.aspx","title":"BMC Chemistry","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"733db058-dfe7-4543-be06-0b67e2f386ba","owner":[],"postedDate":"September 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-09T16:04:53+00:00","versionOfRecord":{"articleIdentity":"rs-7517118","link":"https://doi.org/10.1186/s13065-026-01739-8","journal":{"identity":"bmc-chemistry","isVorOnly":false,"title":"BMC Chemistry"},"publishedOn":"2026-03-08 15:58:26","publishedOnDateReadable":"March 8th, 2026"},"versionCreatedAt":"2025-09-15 11:04:04","video":"","vorDoi":"10.1186/s13065-026-01739-8","vorDoiUrl":"https://doi.org/10.1186/s13065-026-01739-8","workflowStages":[]},"version":"v1","identity":"rs-7517118","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7517118","identity":"rs-7517118","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}